development of dot immunobinding assay … training has been organized which will include both...

Training Manual of ldquoBiosafety issues in the management of genetically modified cropsrdquordquo 1

FOREWORD Agricultural transformation is essential to meet the global challenges of reducing hunger and poverty It faces dual challenges of making food available at affordable price and simultaneously reduces the impact of chemical inputs on the environment An essential aspect of the response to this challenge is to harness all the technologies for a suitable growth in agriculture The transgenic and Molecular Marker Technologies have helped to extend the advantages of conventional plant breeding to meet the increasing need for food in the near future

Genetic Engineering offers seemingly an environmental friendly method of crop protection World now plants about 67 million hectares of transgenic crops mostly resistance to herbicide insect and viruses However several concerns have been raised regarding transgenic crops for pest control such as impact of the transgenic crops on insect density development of resistance effects on the non-target organisms potential for introgression of exotic genes into closely related wild species and Biosafety of the genetically modified food to the human being In response to these concerns four Biosafety centers has been established by Ministry of Environment and Forests (Govt of India New Delhi) under World Bank ndash GEF project for fulfilling the obligation of Cartagena Protocol I am happy to inform that GB Pant University has been identified as one of the institution under capacity building on Biosafety Capacity building on Biosafety related issues are one of the critical elements for the effective implementation of Cartagena Protocol It is proposed to set-up an exclusive National Facility for Biosafety at Pantnagar in Uttaranchal State One of the aims of the centre is to create awareness about the risk associated with the release of transgenic crops in field conditions

The present training on ldquoBiosafety for Monitoring and Evaluation of Transgenic Plantsrdquo is therefore organized to train faculty and scientific personals in north zone agricultural and other universities and research institutions of centre and State agencies This is the first time that such training has been organized which will include both lectures on the current topics related to Biosafety and GMOs testing methods in laboratories Considering Uttarnachal as most vulnerable and ecofragile region this training will also attempts to highlight possible concerns in protecting the biodiversity I wish good luck and success to this training

Dr GK Garg Advisor Biotechnology


PREFACE

Transgenic crops have become reality as then offer great opportunity to move gene(s) across the sexual barrier in more precise and faster ways as well as introgression of traits that would reduce the cost positive environmental impact and enhance the nutritional value Transgenic crops are not a panacea for solving all the problems however there are a number of perceived ecological and economic threats that need to be addressed while producing and deploying transgenic crops for food and nutritional security Considering these threats the training workshop is organized to cater the needs of scientific personals who wish to acquaint themselves about Biosafety related issues in monitoring and evaluation of transgenic crops under field conditions and its impact on biodiversity The main goal of this training is to scintillate the mind of the Scientific Community for realizing the scope and limitations of transgenic technology for sustainable agricultural growth in the country It will cover both relevant lectures and practical concepts of molecular testing methods for evaluation of transgenic crops The training workshop strives to provide a packed module of exercises in systematic manner for on- hands training The faculty members of Department of Molecular Biology and Genetic Engineering namely Dr Sonu Ambwani Dr BR Singh Dr Sneh Narwal Dr Dinesh Pandey Dr Gohar Taj Khan and Research Staffrsquos Mahender Singh Dr Kaushal Gautam who worked round the clock to organize the training workshop and prepare the manual to make it useful to the participants The contributions of non-teaching staffs and contractual workers as Mr AVS Rao Mr RN Singh Mr Nathoo Parsad Mr Abhilakh Singh are gratefully acknowledged Further training workshop will not be possible without visionary leadership of Prof GK Garg Advisor and Coordinator Biotechnology as well as Course Director of present training The painstaking efforts of our guest internal faculty is duly acknowledged The training workshop has been organized under world bank-GEF project and sponsored by Ministry of Environment and Forests The immense interests shown by Department of Biotechnology Govt of India for establishing this National Facility on Biosafety at Pantnagar is duly acknowledged

Dr Anil Kumar Course Organizer Associate Professor Dept of Mol Biol amp Genetic Engg

CBSH GBPUAampT Pantnagar


INDEX

MODULE-1

PLANT GENOMIC DNA ISOLATION FROM LEAF Isolation of high molecular weight genomic DNA from leaf of transgenic plants

MODULE-2

PLANT GENOMIC DNA ISOLATION FROM SEEDS

Isolation of high molecular weight genomic DNA from seeds of BT Cotton transgenic plants

MODULE-3 QUANTITATIVE ANALYSIS OF PLANT GENOMIC DNA

Quantitation of genomic DNA isolated from leaves and seeds of transgenic plants

MODULE-4 AGAROSE GEL ELECTROPHORESIS

Checking the quality and quantity of genomic DNA isolated from transgenic plants by agarose gel electrophoresis

MODULE-5

GEL DOCUMENTATION SYSTEM Demonstration of gel documentation system for visualization of electrophoresed macromolecules (DNA RNA Protein)

MODULE-6

PCR AMPLIFICATION Checking of integration of gene in transformed plants using the powerful technique of polymerase chain reaction

MODULE-7

EXTRACTION OF PROTEINS Extraction of crude proteins from leaves and seeds of transformed plants

MODULE-8

ESTIMATION OF PROTEINS Determination of concentration of crude proteins isolated from leaves of transformed plants by Bradford dye binding methods

MODULE-9

SDS-PAGE ELECTROPHORESIS Separation of proteins isolated from leaves of transformed plants by Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS-PAGE)

MODULE-10

DOT-IMMUNO BINDING ASSAY Development of dot immunobinding assay (DIBA) for detection of expressed protein in transgenic plants


MODULE-11

IMMUNO-DIPSTICK TEST Development of immuno-dipstick test for detection of transgene expression in genetically modified plants

MODULE-12

SANDWICH ELISA Development of indirect ELISA for quantitation of protein expressed by transgenic plants

MODULE-13

SOUTHERN BLOTTING Detection of number of gene copies introgressed in genetically modified plants using the Southern blotting

MODULE-14 RNA ISOLATION AND RT PCR

Detection of transcriptional gene expression in genetically modified plants

MODULE-15 WESTERN BLOTTING

Western blotting for confirmation of transgene expression in terms of protein of desired molecular weight


Contents MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45


MODULE-1

PLANT GENOMIC DNA ISOLATION FROM LEAF Object Isolation of high molecular weight genomic DNA from leaf of transgenic

plants

PRINCIPLE Cells are usually ruptured by mechanical force following several methods The most commonly used method is grinding the plant tissues in mortar and pestle in the presence of liquid Nitrogen Liquid Nitrogen freezes the tissue rapidly and allows fine grinding in mortar and pestle Sometimes plant tissue is ground by using glass steel or tungsten carbide beads in pestle and mortar The disrupted plant cells are lysed and extracted with a suitable buffer which contains a detergent such as CTAB(cetyl trimethyl ammonium bromide ) Heat treatment is often given at this stage to completely lyse the plant cells And after that it is extracted once with chloroform which dissolves most of the impurities like protein carbohydrate cell debris etc Nucleic acids come in aqueous phase which is then separated by centrifugation DNA can be precipitated by addition of ethanolisopropanol and can be spooled out using a glass rod Spooled DNA can be taken in to fresh tube and then it is dissolved in TE buffer and quantitated

Requirements CTAB (N-Cetyl N N N ndashTrimethyl Ammonium Bromide) Tris-

buffer NaCl EDTA Na2 β- mercapto-ethanol (BME) Isopropanol Ethanol

chloroform Isoamyl alcohol

Stock solutions 1 1M Tris-Cl Buffer (pH 80) 100 ml Dissolve 12114 g of Tris-base in 80 ml of ddw Adjust the pH to 80 with 6N

HCl Now make the vol upto 100 ml by ddw Autoclave and store at 40C

2 05M EDTA (pH80) 50 ml Dissolve 93 g of EDTA Na2 in 30 ml of water (add 10N NaOH or NaOH

pallets to adjust the pH80) adjust the final vol upto 50 ml Autoclave and store at

40C

Working solutions 1 Extraction buffer 25 ml 2 (wv) CTAB 05 g

100 mM Tris-Cl 25 ml (1M stock)

14 M NaCl 20475 g

20 mM EDTA 1ml (05M stock)

02 BME 50 μl

2 TE buffer (pH 80) 25 ml

10 mM Tris-Cl 250μl (1M stock)

1 mM EDTA 50 μl (05M stock)

Procedure 1 Wash the leaf material with autoclaved ddw and then with absolute alcohol

2 Grind 05 g leaf material in liquid N2 to fine powder using pre-chilled pestle

and mortar

3 Transfer the powder to 15 ml polypropylene tubes containing 5 ml of pre-

warmed extraction buffer Use spatula to dispense the material completely

4 Incubate samples at 600C for 30 min with occasional mixing by gentle

swirling

5 Add 3 ml chloroform isoamyl alcohol (241) and mix by inversion to

emulsify

6 Spin at 15000 rpm at RT for 10 minutes

7 Remove the aqueous phase with a wide bore pipette transfer to a clean

tube add double volume of isopropanol and mix by quick gentle inversion

8 Keep at -200C for 2-3 hours

9 Spool DNA using a bent pasture pipette amp transfer spooled DNA to another

tube DNA can also be precipated by the tubes spinning at 10000 rpm for

10 min alternatively

10 Wash the DNA pellet in 70 ethanol for 20 minutes

11 Dry the pellet and dissolve in 500 μl TE buffer Store at -200C

Observation

M A

Isolation of DNA from cotton leaves Where M= Marker A= DNA



MODULE-2

GENOMIC DNA ISOLATION FROM SEEDS Object Isolation of high molecular weight genomic DNA from seeds of BT Cotton

transgenic plants

Principal Method described by Benito et al 1992 was employed for the

extraction of DNA from seeds The method is non destructive because only half of

the seeds are used for processing Requirements

Preparation of reagent used 1 Modified DNA extraction buffer 100 ml Tris base 121 g

05 M EDTA 10 ml

SDS 40 g

NaCl 03 g

H2O 65 ml

Adjust pH 80 with concentrate HCl and make volume 100 ml

Autoclave and store at room temp

2 5 M potassium acetate 50 ml Potassium acetate 248 g

H2O 30 ml

Adjust pH 54 with acetic acid and make volume 50 ml and store at room

temperature

3 Isopropanol The isopropanol for extraction should be kept at 00C

4 PhenolChloroform Isoamyl alcohol These were mixed in a proportion of 25241 respectively

5 70 ethanol Ethanol 7 ml

Distilled water 3 ml

Procedure Method described by Benito et al 1992 was employed for the


the seeds are used for processing 1 30 mg of cotton seeds were taken and crushed in pestle and mortar

2 150microl of modified extraction buffer added and left at 650C for 10 minutes

3 60 microl of 5 M potassium acetate was added and vortexed

4 Vortexed solution was left in ice for 20 min

5 Centrifuged in 15 ml micro centrifuge tubes at 15000 rpm for 30 min at

40C

6 Supernatant was transferred to new tube and same volume of chilled

isopropanol was added and kept overnight at 00C

7 Centrifuged at 1000rpm for 15 min

8 The pellet was dried in air and dissolved in 50microl of TE buffer

9 Added 50microl of chloroform-phenol-isoamyl alcohol and tube was gently

shaken

10 Centrifuged at 12000 rpm for 10 minutes at room temperature

11 Supernatant was taken and equal volume of isopropanol was added

12 Incubate the tube at -200C for overnight and centrifuged again at 12000 rpm

for 15 minutes

13 Pellet was dissolved in 70 ethanol and dislodged by tapping


15 Added 100microl TE buffer to dissolve the pellet

Observation


Where M= Marker

A= DNA

M A

Isolation of DNA from cotton seeds

Where M= Marker A= DNA

MODULE-3

QUANTITATIVE ANALYSIS OF PLANT GENOMIC DNA Object Quantitation of genomic DNA isolated from leaves and seeds of transgenic

plants

Principle A reliable measurement of DNA concentration is important for many

applications in molecular biology like complete digestion of DNA by restriction

enzymes and amplification of target DNA by polymerase chain reaction DNA

quantitation is generally carried out by spectrophotometric measurement or by

agarose gel electrophoresis

1A 260 = 50 μgml DNA

Requirements TE buffer quartz cuvette Spectrophotometer DNA

Procedure 1 Take 500 TE buffer in cuvette and calibrate spectrophotometer at 260 nm

as well as at 280 nm

2 Now take 10 μl of DNA + 490 μl TE buffer and mix properly and read the

OD at both 260 and 280 nm

3 Estimate the DNA concentration employing the following formula

A260times 50 times dilution factor Amount of DNA (μgμl) = 1000 4 Judge the quality of DNA from the ratio of the OD values recorded at 260

and 280 nm The A260A280 ratio around 18 (175-185) indicates best quality

DNA Ratio below 18 indicates protein contamination whereas ratio above

18 indicates RNA contamination

OBSERVATION Table Genomic DNA isolated from leafseed Amount of seeds

OD at 260nm

OD at 280nm

Ratio OD260OD280

Yield (microg)



MODULE-4

AGAROSE GEL ELECTROPHORESIS Object To check the quality and quantity of genomic DNA isolated from

transgenic plants by agarose gel electrophoresis

Principle Agarose is extracted from a see weed and is a linear polymer of

different forms of galactose Agarose gels can be casted by melting the agarose in

the presence of desired buffer until a clear transparent solution is obtained The

melted solution is then poured into a mold and allowed to harden Upon hardening

the agarose forms a matrix the density of which is determined by the

concentration of the agarose When an electric field is applied across the gel DNA

which is negatively charged at natural pH migrates towards the anode

Equipments required Horizontal slab gel electrophoresis apparatus procured

from Bangalore genei

Requirements - Tris-base glacial acetic acid EDTA Boric acid Bromo phenol

blue sucrose ethidium bromide

Solutions

Electrophoresis Buffer

1 Tris Acetate EDTA (TAE) 50X solution 1litre Tris base 2420 g

Glacial acetic acid 571 ml

05M EDTA (pH 80) 100 ml

Make the volume up to 1000 ml with ddW Autoclave and store at RT

Working concentration 1X 004M Tris-Acetate 0001M EDTA

or Tris Borate EDTA (TBE) 10X solution 1litre Tris base 108 g

Boric acid 55

05M EDTA 40 ml (pH 80)

Make the volume of the solution up to 1000ml with ddw Autoclave and store

at RT

Working concentration 1X 0090M Tris-borate 0001M EDTA

2 6X Gel loading buffer 025 Bromophenol blue dissolved in 40 (wv) sucrose or glycerol

solution in TE buffer Filter sterilize and store it at 4oC

3 Staining solution Stock solution of Ethidium bromide (10 mgml) Dissolve 10mg EthBr in 1ml

double distilled water Working concentration of EthBr is 05 to 10 μgml

This solution is to be stored at 4oC in dark

Procedure A Gel preparation

1 Take desired amount of electrophoresis grade agarose in 1X

electrophoresis buffer sufficient for constructing the gel If 100ml gel has to

be prepared then for preparation of 08 agarose gel weigh 08g Agarose

amp melted by heating with continuous swirling till a clear solution is obtained

2 Add ethidium bromide to this final concentration of 05 to 10 μgml

3 This molten agarose is poured on the gel casting platform and insert a

comb ensuring that no air bubbles have entrapped underneath the comb

B Loading and running the gel 1 After the gel hardens the comb is withdrawn taking care that the wells do

not tear off The gel tank is filled with sufficient volume of electrophoresis

buffer

2 DNA samples are prepared in 6X gel loading dye and loaded into the wells

with micro-pipette

3 Electrophoresis is carried out at a constant voltage of 50 Volts

C Visualization and photography of DNA in agarose gel 1 DNA gels are visualized on UV transilluminator in gel documentation

system

2 Photograph may also be taken

Steps of performing Agarose gel electrophoresis



OBSERVATION Gel preparation _________ gel Agarose powder ________g

Concentrated buffer _________ml Sample volume ________ul

Run conditions _____min _____volts ______mm Marker migration

Stain _________________________________________

Well number Amount of sample Amount of gel loading dye

Total amount of sample loaded

1 2 3 4 5 6 7 8 9 10


MODULE-5

GEL DOCUMENTATION SYSTEM Object Demonstration of gel documentation system for visualization of

electrophoresed macromolecules (DNA RNA Protein)

PRINCIPLE The Imaging system is powerful digital imaging system ideal for

instant photography of a wide variety of samples The CCD camera

allows imaging of low-light samples in UV-illuminated and fluorescent

applications The instrument is controlled by software which is

designed with ease-of-use in mind Software performs image

analysis and archiving and can prepare images for desktop

publishing

Procedure Start the program

Turn on the computer monitor and (optional) printer Alpha Ease FCTM

software is activated by clicking on the AlphaImager icon This icon can be

moved into the Windows Startup setup to automatically start

(We recommend powering up all system components at once by turning on

the surge suppressor)

If the AlphaImager system software is not running click on its icon in

Desktop

Activate the camera image acquisition controls

In the window title ldquoTool Barrdquo click on the CAMERA icon to activate the

image acquisition software features

In the ldquoImage Acquisitionrdquo Window click on the blue FOCUS button

Use the switch panel controls on the MultiImage cabinet or the identical

lsquovirtualrsquo software controls to activate the Transillumination or Reflective

White light illumination to provide proper illumination to help focus or

position your sample Also choose the appropriate optical filter for your

sample type

Position the sample and optimize the lens settings Position your sample on the preferred illumination source Fluorescence

samples that require epifluorescence or transillumination of UV energy

should be placed on the black UV filter surface For colorimetric samples

such as protein gels films or blots fold down the white light table for your

sample Use the monitor real time readout display to position your sample in

the middle of the image acquisition window


Open the sliding door in the top of portion of the MultiImage FC cabinet to

allow adjustments to the standard zoom lens Adjust for proper focus and

zoom such that the image fills the entire image acquisition window Adjust

the aperture to allow for clear imaging of the sample to allow for proper

focusing Hint Use the wells as a focusing reference

Capture an image Close the cabinet door

Turn on the illumination source (UV or white light) using the touch panel or

lsquovirtualrsquo software controls and click on the green EXPOSE PREVIEW button

Note For UV illuminated samples adjust the lens aperture to full open

Click on show saturation to be sure the sample is within the linear range of

the camera (optional)

Adjust the camera aperture and or exposure time for optimal lighting

Click on the red ACQUIRE button when the image is displayed as desired

Save the original image Click on the Save Image function in the FILE menu or click on the SAVE or

SAVE AS icon in the ldquoTool Barrdquo window

Enter a file name (255 characters or less) and select the directory to which

is should be saved

Specify the file format (TIF BMP PCX MAC Color TGA)

Click OK to save the file

Enhance the display (Optional) Adjust the black white and gamma levels by moving the slider bars at the

right of the image in the ldquoContrast Adjustrdquo window

Apply digital filters found in the ldquoTool Boxrdquo window under ENHANCEMENT

and FILTERS (to stop a filter hit any key on the keyboard to reverse the

effects of a filter click UNDO)

Add text boxes arrows etc to the image using the annotation tools in the

ldquoTool Boxrdquo window under ENHANCEMENT and ANNOTATE

Print the image using the large PRINT button in the ldquoTool Barrdquo window or the pull-

down FILE menu option

Analyze the sample using the analysis features in ldquoTool Boxrdquo ANALYSIS for

quantitative analysis

Fig Gel documentation (Alpha Imager) system



MODULE-6

PCR AMPLIFICATION Object Checking of introgression of gene in transformed plants using the powerful

technique of polymerase chain reaction

Principle PCR DNA amplification is a very simple method for in vitro amplification

of specific nucleic acids using Taq DNA polymerase and minimum two

oligonucleotides specific to the DNA to be amplified The technique involves

repeated rounds of in vitro DNA synthesis The reaction mixture consists of Taq

DNA polymerase DNA template (DNA to be amplified) Taq DNA polymerase

assay buffer forward and reverse primer dNTPs water

The technique is based on three simple steps which are as follows

1 Denaturation of the duplex template into single strands

2 Annealing of primers to each original strand for new strand synthesis

3 Extension of the new DNA strands from the primers This is one cycle of

amplification The amount of amplified product will be 2n copies where n =

no of cycles

Factors affecting amplification 1 Sample volume and microfuge tube

Most amplifications are performed at 20 50 or 100 μl volume in 02 or 05ml

microfuge tubes Large volumes do not allow adequate thermal equilibrium of the

reaction mixture

2 Template DNA

Typically nanogram amounts of cloned template upto μg amount of

genomic DNA or 20000 target copies are chosen to start optimization trails Higher

amounts of template DNA inhibits or results in non-specific amplification

3 Primers They are oligonucleotides ranging from 15-30 bases Primers designed

should be such that at 3prime ends they should not contain more than two bases

complimentary to each other This can result in the formation of primer dimer No

internal secondary structure should be present G+C content typically should be

40-60 Optimal annealing temperatures must be determined empirically The

concentration of primers ranges between 01-1 μM The melting temp (Tm) of both

forward and reverse primers should be same

4 Deoxy nucleotide triphosphates Typically the final conc of each dNTP in a standard amplification reaction

mix is 200 μM It is important to keep the 4 dNTPrsquos concentrations above the


estimated Km of each dNTP (10-15 μM) and balanced for best incorporation

fidelity

5 Taq DNA polymerase buffer The 10X buffer which contains 500 mM KCl 100 mM Tris HCl (pH 90) 15

mM MgCl2 01 (wv) gelatin is supplied It is advisable to carry out a titration

series in small increments over the 15 to 4 mM MgCl2 range to determine the

MgCl2 concentration producing the highest yield of a specific product Too little free

Mg will result in no amplification and too much may produce a variety of unwanted

products

6 Taq DNA polymerase Taq DNA polymerase is a 94 kDa thermostable DNA polymerase which

lacks 3prime to 5prime exonuclease activity but has 5prime to 3prime exonuclease activity in addition

to 5prime to 3prime polymerase activity For most amplification reactions 15 to 2 units of

enzyme are recommended as excess of it leads to non-specific amplification

7 Thermal profiles DNA denaturation is the critical step in the DNA amplification reactions The

time specified for DNA depends on various factors like nature of template GC

content secondary structure etc For most amplifications incubation time for DNA

denaturation is 30 sec-1 min at 92 to 950C with 940C being standard choice

Primer annealing Primer annealing in most of the cases is done 37 to 550C The specified

annealing temperature is calculated empirically and is usually 50C less than the Tm

of the primers used Extreme annealing temperature may result in non specific

amplification

Primer extension Primer extension in most amplifications occur efficiently at a temp of 720C

and seldom needs optimization Time specified for extension depends on the

length of target sequence For example for 1 kb target DNA usually 1 minute

extension is recommended

Reagents supplied 1 Taq DNA polymerase

Supplied 30 units as 3 unitsμl is recommended per reaction Store at -200C

or freezer compartment of fridge

2 Deoxynucleotide triphosphate

3 dNTP mix is supplied as 30 μl and recommended use is 3 μlreaction This

mixture has a final concentration of 25 mM of each dNTP and a 10 mM

concentration of total mix Store at -200C or freezer compartment of fridge

4 Assay buffer

Supplied 100 μl 10X Taq polymerase assay buffer with magnesium chloride

Composition 100 mM Tris HCl (pH 90) 500 mM KCl 15 mM MgCl2 and 01

gelatin Store at -200C or freezer compartment of fridge

5 DNA template

This is a genomic DNA purified from Serrratia marcescens of concentration

200 ngμl 10 μl is supplied use 1 μl reaction

6 Control primers

Forward primer It is 18 bases long oligomer Use 1 μl per reaction supplied 10 μl

of 100 ng μl concentration

Reverse primer It is 28 bases long oligomer Use 1 μl per reaction supplied 10 μl

of 100 ngμl concentration

7 Nuclease free H2O Supplied at 1ml store at 40C

8 Mineral oil Supplied at 05ml store at RT

9 Agarose 5g store at RT

10 Gel loading dye 100 μl store at 40C

11 PCR tubes

12 Contol DNA marker 5μg

13 50X TAE 40ml

Procedure DNA amplification For the reaction add the following reagents to a PCR tube

10X Assay Taq Pol Buffer + 15 mM MgCl2 5 μl

dNTP mix 3 μl

Template DNA (200 ngμl) 1 μl

Forward primer (250 ngμl) 1 μl

Reverse primer (250 ngμl) 1 μl

Taq DNA polymerase (3 uμl) 1 μl

Sterile water 38 μl

Total reaction volume 50 μl


Mix the contents gently and layer the reaction mix with 50 μl of mineral oil

(optional as per the specification of PCR machine)

Carry out the amplification using following the reaction conditions for 30 cycles

Step Temperature Time

Initial denaturation 940C 1 min

Denaturation 940C 30 sec

Annealing 480C 30 sec

Extension 720C 1 min

Final extension 720C 2 min

1 After the reaction is over take out the reaction mix and run 10 μl of the aq

layer in 1 agarose gel for 1 to 2 h at 100 volts Run the sample along with

marker and locate the amplified product by comparing with the 08 kb

fragment of the marker

2 Stain with ethidium bromide and visualize under UV light for 08 kb DNA

fragment

Observation

PCR amplification and detection of different gene families (Lane M 100 bp ladder Lane 1-12 PCR of different gene families Lane M Marker lambda EcoRI Hind III double digest

Fig PCR Thermocycler



MODULE-7

EXTRACTION OF PROTEINS Object Extraction of crude proteins from leaves and seeds of transformed plants

Principal Plant cells are mechanically lysed by grinding the tissue or leaves in

mortar and pestle in presence of liquid N2 Leaf protein is thn solubilized in

Tris buffer and cellular debris is removed by repeated centrifugation

Protocol I Extraction of crude proteins from leaves

Requirements Mortar and pestle (autoclaved) ice ice bucket centrifuge tubes

Extraction buffer (100 ml) 02 M TrisCl (pH = 80)

Procedure 1 Weigh 1 gram of plant leaves

2 Transfer the leaves in to mortar and add 5 ml of 02 M Tris-Cl (pH=80)

3 Keep the mortar in ice bucket and crush the leaves with the help of pestle

for 20 min till a fine slurry is made

4 Transfer the slurry in centrifuge tubes and centrifuge at 10000 rpm at 40C

for 20 min

5 Transfer the supernatant in fresh tubes If supernatant has some particulate

matter or debris centrifuge again at 10000rpm for 5 min

6 Store the supernatant at 40C till further use and quantification

Protocol II Extraction of crude proteins from Bt and Non Bt cotton seed


1 Extraction Buffer PH (72) (100ml) 25mM Na Phosphate

50mM Nacl (5M) 5mM EDTA (05mM) 2 PMSF (01 M Stock in ethanol) Procedure 1 One gram Bt and Non Bt cotton seed crushed with Glass beads for 20 minute

and further crushed with 5 ml protein extraction buffer for 10 minutes 2 Centrifuge at 10000 rpm at 4degC for 20 minutes 3 Take supernatant and add PMSF to final concentration of 20mM 4 Store the protein samples at -20degc

MODULE-8

ESTIMATION OF PROTEINS Object Determination of concentration of crude proteins isolated from leaves amp

seeds of transformed plants by Bradford dye binding methods

PRINCIPLE

The Bradford assay is a very popular protein assay method because it is

simple rapidinexpensive and sensitive The Bradford assay works by the action

of Coomassie brilliant blue G-250 dye (CBBG) This dye specifically binds to

proteins at arginine tryptophan tyrosine histidine and phenylalanine residues

CBBG binds to these residues in the anionic form which has an absorbance

maximum at 595 nm (blue) The free dye in solution is in the cationic form which

has an absorbance maximum at 470 nm (red) The assay is monitored at 595 nm

in a spectrophotometer and thus measures the CBBG complex with the protein

The Bradford dye assay is based on the equilibrium between three forms of

Coomassie Blue G dye Under strongly acid conditions the dye is most stable as a

doubly-protonated red form Upon binding to basic and aromatic amino acid

residues of proteins however it is most stable as an unprotonated blue form

Requirements Comassie Brilliant Blue (CBB) G-250 Ethanol ortho- Phosphoric

acid BSA double distilled water Funnel filter paper test tubes

Solutions 1 Bradford dye

Dissolve 100 mg CBB G-250 in 50 ml ethanol add 100 ml ortho- phosphoric

acid (85 wv) Make the volume up to 1 lt with double distilled water Filter

the solution and store at 40C in dark coloured bottle The solution prepared

should be used within 2 weeks

2 BSA standard Stock solution 1 mgml


1 Take 12 glass test tubes having 5 10 20 40 60 100 μl of BSA in

duplicate Add extraction buffer double distilled water to make volume up to

300μl Prepare the samples and blank as per following table


BSA stock solution Extraction bufferdouble distilled water

Total Volume (μl)

5μl 10μl 20μl 40μl 60μl 100μl Blank

Protein sample 1(20 μl) Protein sample2 (40 μl)

295μl 290μl 280μl 260μl 240μl 200μl 300 μl 280μl 260μl

300μl 300μl 300μl 300μl 300μl 300μl 300μl 300μl 300μl

2 Add 3 ml of dye in each tube

3 Incubate them for 5 min at RT 370C

4 Now read the absorbance of all samples at 595 nm in colorimeter

5 Draw a standard graph between the BSA concentration and absorbance

6 Calculate the amount of protein in the unknown sample with the help of

graph

OBSERVATION BSA concentration (microgml) OD at 280 nm Unknown protein sample

MODULE-9

SDS-PAGE ELECTROPHORESIS Object Separation of proteins isolated from leaves of transformed plants by

Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS-PAGE)

PRINCIPLE The separation of macromolecules in an electric field is called electrophoresis In an early form of electrophoresis dissolved protein mixtures were placed in a U-shaped buffer-filled channel and subjected to an electric field Resolution was poor and any disturbance of the apparatus compromised the separation Gels were developed to serve as solid supports for electrophoresis so that the separated products remain separated and can be easily stained and handled The development of the stacking gel which compresses the sample into bands a few micrometers thick added a major improvement to the resolution of gels Other landmark improvements to protein electrophoresis were the use of polyacrylamide for control of separation by molecular size and the use of sodium dodecyl sulfate (SDS lauryl sulfate) to denature proteins in order to ensure reproducibility of the technique SDS is an anionic detergent meaning its molecules have a net negative charge It binds to most soluble protein molecules in aqueous solutions over a wide pH range Polypeptide chains bind amounts of SDS that are proportional to the size of the molecules The negative charges on SDS destroy most of the complex (secondary and tertiary) structure of proteins and are strongly attracted toward an anode (positively-charged electrode) in an electric field

A polyacrylamide gel with acrylamide content above a critical density restrains larger molecules from migrating as fast as smaller molecules Because the charge-to-mass ratio is nearly the same among SDS-denatured polypeptides the final separation of proteins is dependent almost entirely on the differences in molecular weight (MW) of polypeptides In a gel of uniform density the relative migration distance of a protein (Rf) is negatively proportional to the log of its MW If proteins of known MW are run simultaneously with the unknowns the relationship between Rf and MW can be plotted and the MWs of unknown proteins determined Protein separation by SDS-PAGE is used to determine the relative abundance of major proteins in a sample their approximate molecular weights and in what fractions they can be found The purity of protein samples can be assessed Different staining methods can be used to detect rare proteins and to learn something about their biochemical properties Specialized techniques such as Western blotting two-dimensional electrophoresis and peptide mapping can be used to detect extremely scarce gene products to find similarities among them and to detect and separate isoenzymes ofproteins

Requirements Acrylamide bis- acrylamide SDS Tris-HCl Tris-base Glycin β-

mercaptoethanol (BME) Bromophenol blue Glycerol Comassie brilliant blue R-



250 TEMED Ammonium per sulphate (APS) EDTA glacial acetic acid and

methanol

Solutions 1 30 Acrylamide amp bis-acrylamide stock solution 292 g of acrylamide

and 08 g of N Nrsquo- methylene bis acrylamide dissolved in 100 ml of ddw Stir

at magnetic stirrer and warm it slightly so as to dissolve it completely Filter

and Store at 40C in a dark coloured bottle

2 10 SDS 10 g SDS was dissolved in 100 ml of double distilled water Keep at 370C or

warm it slightly for complete dissolution and store at RT

3 Stacking gel buffer 05 M Tris-HCl (pH 68) Autoclave amp store at 40C

4 Separating gel buffer 15 M Tris-HCl (pH 88) Autoclave amp store at 40C

5 Electrophoresis buffer (pH 83) (Tris-glycine buffer) Tris base 25 mM

Glycine 192 mM

SDS 01

6 Sample buffer(2X) Tris-HCl (pH 68) 62 mM

SDS 2 Glycerol 10 BME 5 EDTA 01 BPB 01

7 Staining solution 50 methanol

10 glacial acetic acid

01 CBB - R250

8 Destaining solution 10 methanol


9 10 Ammonium per sulphate 01gml It should be prepared fresh 10 Composition of 12 separating gel (20ml)

30 Acrylamide stock 80ml

Separating gel buffer 50ml

10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml

11 Composition of 5 stacking gel (8ml) 30 Acrylamide stock 13ml

Stacking gel buffer 20ml

10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml

APS and TEMED are added just before pouring of the gel solution as

TEMED initiates the cross-linking of the gel

Procedure (A) Casting the gel

1 Carefully clean the glass plates with warm soapy water Rinse them

thoroughly with ddw Dry and wipe them with ethanol

2 Assemble the gel plates with the spacers and the clamp Now pour the

separating gel solution prepared between the glass plates Overlay it with

water and allow it to polymerize for 30 ndash 40 min at 370C

3 Remove the overlay solution Wash the gel 2-3 times with ddw Now pour

5 stacking gel prepared Insert the comb and let the gel again polymerize

4 Remove the comb clean the wells with a syringe and assemble the gel

plates in the electrophoresis chamber filled with the electrophoresis buffer

Pre run it at 80V for 15min

(B) Sample preparation amp running the gel

1 Approx 50μg of protein sample is taken with 2x sample buffer in 11 ratio

The mixed samples are boiled in water bath for 5 min

2 Centrifuge the samples at 10000 rpm for 5 min

3 Load the samples with the help of micropipette

4 Run the gel at 2-3 mAlane at constant voltage of 50 Volts till the sample is

in stacking gel and after that the voltage can be raised to 80 or 100 volts

Allow the gel to run till the dye front reaches 05 cm from the lower edge of

the gel

5 After completion of electrophoresis gel is taken out in a tray containing the

dye solution

6 Leave the gel in staining dye solution ON

7 Next day gel is taken out and kept in destaining solution 2-3 changes of

destain are given The gel is destained till the bands become visible

Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein

Steps of performing SDS-PAGE electrophoresis



MODULE-10

DOT-IMMUNO BINDING ASSAY Object Development of dot immunobinding assay (DIBA) for detection of

expressed protein in transgenic plants Principle This assay is based on the adsorption of proteins onto membranes and

quantitation of the chromogenic product that precipitated in the membrane by

visual examination or measurement by automated equipment The design of a Dot

Blot assay requires preparing an enzyme- labeled antibody (or antigen) which

binds to a membrane bound antibody (or antigen) In a subsequent step bound

molecules are separated from unbound reactants In the Dot Blot assay antigen is

first adsorbed on a membrane surface The membrane is soaked in a ldquoblocking

bufferrdquo to saturate the membrane with nonspecific protein After a wash to remove

excess blocking proteins the membrane is soaked in a solution of antibody-enzyme

conjugate to bring about the formation of the enzyme linked-antibody-antigen

complex Washing the membrane by soaking in an aqueous buffer is the method

used to separate bound from unbound reactant When the membrane is soaked in

a substrate solution the enzymatic reaction produces an insoluble product which

precipitates as a circular dot on the membrane

Requirements Reagents

PBS Phosphate buffer saline (015M pH 75)

Blocking Solution PBS + 025 BSA

Procedure Dot blot is an efficient technique for detection of proteins for it is very

rapid technique and also semi quantitative in nature

1 Wash the Nitrocellulose membrane in sterile distilled water amp dry it and

make the imprint by dot blot apparatus

2 2μl aliquots of sample containing required 1μg of antigen (crude protein

extract of transgenic plants) was spotted on to the glossydull surface of

nitrocellulose membrane and allow it drying for 15 min

3 Treat the nitrocellulose with 20 blocking solution for 2 hr

4 Wash the nitrocellulose blot thrice with PBS and 025 BSA for 5min

5 Membrane is incubated with required dilution of 1st antibody (1500) diluted

in dilution buffer at room temperature for 2hr

6 Wash the nitrocellulose blot thrice (5min each) in the PBS + 025 BSA

7 Membrane is incubated with alkaline phosphatase conjugated secondary

antibody for 1 hr antibody (diluted to 11000 in antibody dilution buffer)

8 The membrane is washed 3 times (5min each) with the PBS+025 BSA

9 Alkaline phosphatase (2ml) colour development substrate solution ie

BCIPNBT is added and incubated for 10 min

10 The reaction is stoped by adding distilled water and by removing the

substrate

PBS (1L)

Observation

(Dot Blot immune assay )



MODULE-11

IMMUNODIPSTICK TEST Object Development of immunodipstick test for detection of Bt transgene

expression in genetically modified plants

Principle The dipstick test was developed for the detection of the protein

expression of introgressed gene in transgenic plants These dipsticks were

screened for reactivity of various expressed proteins for various agronomic traits In dipstick test the anti FC antibody is tagged by an enzyme and the

antigen is coated on a solid support The first antibody raised against desired

protein to be expressed in transgenic plants react with expressed protein and this

antigen ndashantibody complex subsequently bind with second antibody coupled with

enzyme The enzyme produce colour products on addition of the substrates and

this are only possible when first antibody bind to the solid support The

development of color indicates the antigen-antibody reaction

Requirements Bt-Express kit

Materials Extraction buffer Procedure

1 Pick up a cotton seed or a piece of leaf tissue (two discs of about 10 cm

diameter each) and break it open

2 Take the white coloured internal embryo matter and transfer it into the vials

provided

3 Add 05 ml (marked on the vial) of sample extraction buffer provided

4 Crush the embryoleaf disc using the pestle provided with the kit

5 Dip the Cry 1 Ac Bt instant-check strip into the vial to take care that only the

end marked as sample in dipped into the sample Wait until the sample

solution (purple coloured) travels till the top end of the strip and the filter pad

at top-end is almost completely wet This should take about 15-20 minutes

6 If one band develops half way through eh strip along with another at the top

(two bands) It indicates presence of Cry 1 Ac in the sample It is a positive

Bt- cotton sample

7 If only one band develops at the top of the strip it indicates that the sample

is negative for Cry 1 Ac and is a non- Bt sample

PRECAUTIONS

1 Clean the pestle thoroughly before re use Any minor contamination of the

sample with a positive sample can lead to false positive

2 Keep the strips in airtight pack in reingerated condition to ensure sustained

activity

3 If unused the strips may lose activity on prolonged storage of 6 months

4 Do not keep strips outside the seal pack In a humid environment the strips

absorb moisture on prolonged exposure and have a slow flow rate

5 Remove the strips from the sample after 20-25 minutes to avoid a possible

reverse flow and appearance of false positives

6 Leaves of Bt-plants older than 90 days have less Cry 1 Ac hence the

sample line intensity is usually very light It is recommended that the strips

not be used on such samples

7 Sometimes if the sample extract is coloured or concentrated there may be

a light grey coloured band in the sample line region This should not be

considered positive

Observation

Immunodepistic assay for detection of cry protein in the protein sample extracted from 1 gm cotton seed by using three different extraction procedures ie AB and C



MODULE-12

SANDWICH ELISA Object Development of sandwich ELISA for quantitative detection of Bt protein

expressed by transgenic plants

Principle In sandwich ELISA trapping antibody is coated on a solid support such

as polystyrene microtitre plates Antigen (protein sample) is added and a second

antibody which is tagged with an enzyme is used to detect the antigen The

enzyme produces coloured products on addition of the substrate and this is

possible only when antibody binds to the antigen Thus the development of colour

indicates the antigen antibody reaction

Requirements DesiGen QL 96 ELISA Plate kit (Full) for cry1 Ac cry1 Ab

(DGH030)

The desigen cry 1 Ac plate is designed for qualitative (presenceabsence)

laboratory detection of cry 1 Ac protein in cottonseed or cotton leaf tissue samples

by sandwich ELISA

Materials provided

Antibody-coated 96 well plate

Anti-Cry 1 Ac conjugate

1 ppb Cry 1 Ac control

10x Sample Extraction Buffer

10x buffer A

20x Substrate Buffer

Stop solution

Solutions

a) 1x Sample Extraction Buffer Take 50 ml of 10x sample extraction buffer and dilute it to 500 ml by adding

ddw use 500 μl per sample Store at 40C

b) 1x buffer A (wash buffer) Take 100 ml of 10x buffer A dilute it to 1L by adding DW

c) 1x substrate buffer (Should be prepared freshly before use)

For one plate Take 500 μl of 20x substrate provided add 95 ml of

deionized water to make it to 10 ml Mix well and add 100 μl well of this

substrate


Sample preparation

For seed extracts Imbibe cotton seeds overnight in water Remove seed

coat and cut each seed to be tested in half with a clean blade Place one half of the

seed in a micro-centrifuge tube and add 500 μl 1x sample extraction buffer Crush

with a pestle for 30 seconds Use 100 μl of this extract for sampling

For leaf extracts punch out 2-3 leaf discs with a mcf tube by placing a leaf between

the lid and the tube opening and closing the lid onto the leaf Add 500-μl 1x sample

extraction buffer Crush with a pestle for 30 seconds Use 100 μl of this extract for

sampling Note avoid cross-contamination between samples

Procedure

1 To the pre-coated plate add 100 μl well of anti-cry 1 Ac conjugate

2 Add 100 μl of the sample extract and 1ppb positive control provided to the

appropriate wells and incubate the plate at room temperature for 45 min

3 Wash the plates four times with 1x buffer a (wash buffer) with each washing

of 5 min Pat the plates dry (wells down) on blotting paper to remove excess

buffer

4 Add 100 μl of freshly prepared 1x substrate per well (Take care not to touch

the wells while adding the substrate)

5 Incubate the plate at room temperature in dark for 15 min Add 100μl well

stop solution after 15 min of incubation

6 Measure the absorbance at 450 nm using an ELISA plate reader The

absorbance of a blank well must be subtracted from absorbance values of

samples and controls

Observation

Microtiter ELISA developed in 96 well plate


MODULE-13

SOUTHERN BLOTTING Object Detection of number of gene copies introgressed in genetically modified

plants using the Southern blotting

Principle This procedure is used for hybridizing labeled ndash DNA probes to DNA

fragments separated on an agarose gel and then blotted on nitrocellulose (or

nylon) membrane Southern blot analysis involves the following steps

a) Restriction (digestion) of DNA Plant DNA is restricted using restriction endonucleases The restriction

endonucleases used are type II enzymes which recognize either 4 5 6 or 8 bp

long sequences and generate either 3rsquo protruding ends 5rsquo protruding ends or blunt

ended fragments The next step is the separation of restricted fragments using


b) Agarose gel electrophoresis Agarose gel electrophoresis is used to separate DNA molecules according

to their size The negatively charged DNA molecules migrate in an electrical field

from negatively charged cathode to positively charged anode with smaller

molecules migrating faster than the bigger ones through the pores of the matrix

The pores of the matrix can be changed by altering the agarose concentration

After electrophoresis DNA fragments are visualized by staining with ethidium

bromide which fluoresces in UV

c) DNA transfer to nylon membranes DNA hybridization requires the breakage of hydrogen bonds between the

complementary strands of DNA which can be achieved either by high temperature

treatment or treatment with denaturing agents like formamide These treatments

cannot be done on agarose gels Hence DNA is transferred from the gel onto a

synthetic membrane Nylon coated with nitrocellulose is used which combines the

physical strength of nylon membrane with high resolution of nitrocellulose

membrane The banding pattern is preserved during the transfer The weak

interaction between the DNA and the membrane achieved during transfer is further

modified into a covalent bond by heating to 80oC


Solutions Reaction Mixtures a) Restriction of DNA 1 Mix the following in a sterile 15ml Eppendorf tube

DNA - 5 μg

10x buffer - 50μl

Restriction enzyme - 25μl (10unitsμl)

Sterile water to - 50μl

2 Incubate the mixture at 37oC for 5 hr or overnight

3 Add 5μl of 10x days to the sample centrifuge for 5 sec in an Eppendorf

centrifuge and load on to an agarose gel

b) Agarose gel electrophoresis 1 10x TBE buffer

2 Gel loading buffer (10x dye) c) DNA transfer to nylon membrane 1 025 M HCl Concentrated HCl - 215ml H2O - 9785ml

2 Denaturing Solution (1 litre) (15M NaCl and 05M NaOH) NaCl - 8766g NaOH - 200g Add ddw - to 1 litre

3 Neutralizing solution 1M Tris-Cl (pH 80) + 15M NaCl Tris base - 12112g NaCl - 8766g Adjust pH to 80 with HCl and make up the volume to 1 litre with ddw and

autoclave before use

4 20x SSC (3M NaCl + 03M Sodium citrate pH 70) NaCl - 17532g Na Citrate - 88213g ddw - to 1 litre

Adjust pH to 70 with citric acid and make up the volume to 1 litre with ddw

and autoclave before use

PROTOCOL a) Agarose gel electrophoresis (08)


1 Weigh 08 g agarose and put in a 250 ml conical flask Add 100ml of 1x TBE

buffer and gently boil the solution in a microwave oven with occasional mixing until

all agarose particles are completely dissolved Allow it to cool to 500C Add 10 μl of

ethidium bromide (10mgml) and mix well Prepare the gel mould and keep the

comb in position Pour the cooled gel solution into the gel mould and allow the gel

to set for 20min

2 Fill the horizontal electrophoresis chamber with 1x TBE Remove the comb

from the gel and place gel with the tray in the electrophoresis chamber 3 Load the digested DNA sample carefully into the wells In one well load a

standard marker (Lambda DNA restricted with HindIII) 4 Run the gel at 20mA overnight 5 Visualize the DNA bands on a UV transilluminator and place a ruler next to

the gel to be able to determine the fragment sizes later on Take a picture using

Polaroid camera with a red filter Problems and Remedies Digestion Problem 1 Endonuclease or exonuclease contamination

Remedy 1 Titrate enzymes properly switch to cleaner enzymes changes

suppliers clean sample as described above

Problem 2 Partial digestion

Remedy 2 Use more enzyme two step digestion switch to better enzyme

change supplier clean up DNA (as above) realize to remove excess

salt

Electrophoretic artifacts Problem 3 Retardation due to excess DNA

Remedy 3 Use less DNA use purified or semi purified organellar DNA

Problem 4 Smiling bands

Remedy 4 Remove any bubbles from wells increase run times to decrease

effects of differential heating

Problem 5 Fuzzy bands

Remedy 5 Reduce buffering capacity of running buffer (make new buffer)

Problem 6 Missing small bands

Remedy 6 Reduce electrophoresis time or use a combination of agarose and

polyacrylamide gels

Problem 7 Missing large bands

Remedy 7 Too much BSA in digests could also be due to DNA degradation


Problem 8 Non-specific background (ie flecking) in gels loaded with end-

labelled samples (particularly agarose gels)

Remedy 8 Use higher grade agarose making certain it is completely dissolved

make sure plates and apparatus are clean rinse gels before drying

down

b) Transfer of DNA to nylon membrane 1 After staining and photography of the gel dip and shake it in 200 ml of

025M HCl for 10-15 min at room temperature in a glass baking dish until

the bromophenol blue barely turns yellow

Note Acid depurinates the DNA breaking large fragments into smaller

pieces for more efficient transfer

2 Decant the HCl and rinse with distilled H2O for 1 min and denature the DNA

by soaking the gel in several volumes of denaturing solution for 1 hr at room

temperature with constant shaking 3 Decant the denaturing solution rinse the gel with distilled H2O of

neutralizing solution for 1 hr at room temperature with constant shaking 4 Wrap a piece of Whatman 3 MM paper around a piece of glass plate and

place it inside a large baking dish Fill the dish with 20x SSC almost to the

top and smooth all air bubbles in the 3MM paper with a glass rod 5 Invert the gel so that its original underside is now upper most and place it on

the wet 3 MM paper Remove air bubbles if any between the gel and the

paper 6 Cut a piece of nylon membrane about 1-2 mm larger than the gel Dip it in

sterile water (20 min) and then float the membrane in 20x SSC for5-10 min 7 Place the wet nylon membrane on top of the gel and remove the air bubbles

that are trapped between the gel and the membrane Note Air bubbles trapped between the gel and membrane cause uneven or

incomplete transfer

8 Wet two pieces of Whatman 3MM paper cut to exactly the same size as the

gel in 20x SSC and place it on top of the nylon membrane followed by 6 cm

stack of dry paper towels Put a glass plate on top of the stack and weigh it

down with 500g weight

Note Do not touch nylon membrane with naked hands Always wear gloves

and use forceps while handling nylon membranes Ethidium bromide is

carcinogenic hence wear gloves when using it or whenever ethidium

bromide gels are handled


9 Allow transfer of DNA to proceed for 6 hr to overnight Small fragments of

DNA (lt1 kb) transfer from a 08 gel within an hour or two while transfer of

DNA (gt15 kb) takes 15 hr or more

10 Disassemble the blot in reverse order and using a soft pencil clearly label

the slots on the membrane Also mark the filter to define its orientation

relative to the gel

11 Soak the membrane in 10x SSC at room temperature for 5-10min

12 Place the filter on 3MM paper to air dry

13 Bake the membrane at 80oC for 1 hr in a vacuum oven

14 Soak the membrane in 2x SSC seal it in plastic bag and store in the dark in

refrigerator until use

c) Preparation of probe by oligolabelling (Using Hexalabel DNA labeling kit- MBI fermentas)

The method relies on the priming of the polymerase reaction on the

template DNA with random hexanucleotide primers The complementary strand is

synthesized from the 3rsquo end of the primer with the help of the large fragment of

DNA polymerase I Exonuclease Minues (Klenow Fragment exo) in the presence

of labeled deoxyribonucleoside triphosphates

Detailed Protocol for New Users A Important Information for All users Please read all sections of this manual before proceeding with any

experiments

Nucleic acids cannot be quantitated by absorbance at 260 nm after Psoralen-

Biotin labeling because Psoralen absorbs at this wavelength interfering with an

accurate reading and potentially destroying the cross links that connect biotin

to the nucleic acid It is recommended that quantitation is done before labeling

with Psoralen-Biotin Yield from the procedure will be 90-100 with a slight loss

resulting from the butanol extraction

Psoralen-Biotin interacts preferentially with T residues in DNA and U residues

in RNA It also interacts with C residues Because of this the efficiency of

labeling small (lt100base) polynucleotides varies with the proportion of U T

and C residues and may be affected by their context

Use only nuclease free water for rinsing glassware If necessary use a

nuclease decontamination reagent (such as RNase ZapTM) for cleaning

glassware before rinsing with water


Try the labeling reaction using the Unlabeled control DNA at least once This

will help you to interpret your results and will also ensure that the reagents are

performing according to specifications

Thin walled PCR tubes are ideal for heat denaturing and quick-chilling nucleic

acids before labeling Very rapid chilling of nucleic acids after heat denaturation

is essential to prevent them from e-annealing This can be achieved by placing

the rubes in powdered dry icemethanol in liquid nitrogen or in ice water

B Sample purity and concentration Almost any nucleic acid can be labeled with this kit (eg PCR product

cDNA linearized plasmid in vitro transcript etc) Oligonucleotides as small as 47

mers have been successfully labeled using Psoralen-Biotin The procedure below

gives instructions to label 5-500 ng of sample in a 10μl reaction (5 ng is not

enough labeled nucleic acid for most applications) The reaction can be scaled up

proportionally to accommodate more nucleic acid if desired We have labeled up to

100μl of sample in a single well of a 96 well plate without observing any negative

effect on labeling efficiency For volumes over 100μl we recommend dividing the

sample into 2 or more wells in the 96 wells plate for the labeling reaction

I Sample purity There should be no free nucleotides in the preparation (eg from PCR) If the template is a PCR product remove the free nucleotides with a spin

column or with 2 successive isopropanol precipitations before attempting to label it

using Psoralen-Biotin

RNA sample must be free of DNA eg transcription template To remove DNA treat the RNA with DNase followed by Proteinase K SDS

digestion and acid phenol extraction (procedures 1-3 on page 14) finally

precipitate the RNA with ammonium acetate and ethanol Alternatively you can

use Ambionrsquos DNA freeTM DNase treatment and Removal Kit to simplify this

process substantially

Samples must be free of proteins Remove proteins with a proteinase KSDS digestion followed by

phenolchloroform extraction and precipitation Nucleic acid solutions should be

free of phenol or alcohol contaminants the pH should be between 25 and 10 and

the salt concentration should be les than 20mM If the sample does not meet these requirements precipitate it and

resuspend it in TE buffer to 50ngμl


2 Sample concentration

Sample concentration should be 05 ndash 50 ngμl

If your sample is a precipitated nucleic acid dissolve it in TE Buffer to a

concentration of 50ngμl

If the solution is too concentrated dilute it with the TE buffer provided If it is too

dilute concentrate it by precipitation

C Nucleic Acid Labeling 1 Set up a 96 well plate in an ice bath

Place a clean untreated (non-coated) 96-well plate on an ice bath If necessary

wash the 96-well plate with RNase Zap to remove any nucleases rinse with

dH2O and air dry

2 Denature sample at 1000C for 10 min We recommend using thin wall (PCR) tubes for heat denaturation and

cooling because the tube contents can be cooled more quickly than in

ordinary microfuge tubes

Heat 10μl (5-500ng) of the sample in a microcentrifuge tube for at least

10minutes in a boiling water bath or heat block at 1000C Samples that are

not in TE should be in a solution with at least 1mM EDTA so that they donrsquot

degrade during the heat denaturation

If a large volume of nucleic acid solution is being heated it may be

necessary to set the heat block to about 1100C to ensure complete

denaturation

3 Immediately quick- chill the sample by placing in a dry icealcohol bath Extremely rapid cooling is critical for efficient labeling

Liquid nitrogen or an ice water bath may be used instead of dry icealcohol

The sample will freeze simply leave it frozen until you are ready to add the

Psoralen-Biotin in step 5

If you use an ice water bath it must be a slush and not simply ice alone Step 4-6 must be done in reduced light A dim room will suffice If desired

the lights can be completely turned off after the reaction is set up

4 First time use of kit dissolve the psoralen-Biotin in 33μl

dimethylformamide Centrifuge the vial containing the lyophilized Psoralen-Biotin for ndash15 second

at 7000 xg


In dim light reconstitute the Psoralen Biotin in 33μl of DMF that is provided

with the kit Piper up and down a few times to get the Psoralen- Biotin into

solution The solution should be stored at 4oC in the dark

5 Add 1μl Psoralen-Biotin to 10μl nucleic acid sample

Thaw the sample by rolling it between gloved hands As soon as it is

thawed add 1 μl Psoralen ndash Biotin and mix well

Transfer the reaction to a well in the 96-well plate Alternatively the

Psoralen-Biotin can be added to the nucleic acid already in a well of the 96-

well plate Mix the solution with a pipette tip

If the sample volume is gt10μl use proportionally more Psoralen Biotin For

example a 50μl sample would require 5μl Psoralen-Biotin

6 Irradiate at 365 nm for 45 min Place a 365 nm UV light source on the plate directly over the sample and

irradiate for 45 minutes (The dual wavelength hand held UV lamps found in

many labs are suitable Make sure the lamp is set to 365 nm and not to 254

nm) We have not encountered any degradation or crosslinking of nucleic

acids from these UV treatments

Important Medium wavelength (312 nm) and short wavelength (254nm) lamps should

not be used Also be sure that nothing is between the sample and the light

source The irradiation will be compromised if there is plastic warp or glass

between the samples and the UV light By the same token samples cannot

be irradiated through the bottom of the 96-well plate eg on a

transilluminator The distance between the lamp source and the sample

should be lt2 cm

The biotinylation is now complete so for subsequent steps it is not

necessary to shield samples from ambient light

7 Dilute the sample to 100μl by adding 89μl TE buffer

This is done in order to avoid any sample loss during subsequent extraction

with n-butanol Transfer the sample from the 96-well plate to a clean microfuge

tube

8 Extract twice with 200μl water saturated n-butanol

Remove non-crosslinked psoralen-biotin by butanol extraction as follows

a Add 200μl of Water-saturated n-Butanol (shake the butanol well before use

and use the top layer)

b Vortex well and microcentrifuge for 1 minute at 7000 x g


c The butanol layer will be on top piper it off and do a second extraction

Remove as much of the n-Butanol as possible

The traces of n-butanol that may remain (notice cloudiness when the

sample is stored in a refrigerator) will not interfere with future uses of the

labeled probe

The probe is now ready for use

9 Extract with diethyl ether Trace amounts of n-butanol can be removed by extracting with 2 volumes of

water saturated diethyl ether and pipetting off the diethyl ether

10 Storage of biotinylated nucleic acids Store biotinylated nucleic acids at ndash20oC for short term storage (up to a few

weeks) and at ndash80oC for long term storage (months to a year)

To avoid repeated freeze thawing large amounts of labeled probe should

be stored I the dark in small aliquots at ndash80oC The currently used aliquot

maybe stored at ndash20oC The labeled nucleic acid should be stable for at

least one year at ndash80oC in the absence of nuclease contamination

D Using psoralen-Biotin labeled probes 1 Concentration for use in blot hybridization Standard hybridization buffers

Use 01nM final concentration of biotinylated probe in standard Northern

Southern dot blot or colony hybridization buffer

Ambionrsquos ULTRAhybTM

The probe concentration that should be used in Ambionrsquos ULTRAhybTM buffer depends on the probe type

bull RNA probes 01nM (-10ngml of a 300 nt probe)

bull DNA probes 1pM (-01 ngml of a 300 nt probe)

2 Heat denaturation of DNA probes dsDNA probes must be denatured by heat treatment before use in

hybridization applications It is advisable to re-denature probes with each

use

Dilute the probe ndash 10fold with 10mM EDTA (use a minimum of 50μl)

Incubate the diluted probe at 90oC for 10 min

II BioDetect Protocol A When to Use BioDetect


Biotinylated nucleic acids must be bound to positively-charged nylon membranes

before beginning the detection procedure This is typically achieved by

hybridization of the probe with a target that is cross-linked to the blot

Blot hybridizations (eg Northern or Southern blot) Northern or southern blots (preferably using Bright Star-Plus membrane from

Ambion) on positively charged nylon membrane can be directly processed Be

sure to complete the post-hybridization washes before starting the BioDetect

procedure To control background it is important to keep the membrane damp

after the post-hybridization wash steps Background may be higher using

membranes that have been allowed to dry

Gel separated probe (eg RPAs S I Nuclease Assays) The transfer of polyacry1amide gel-resolved probes to the Bright- Star-Plus

membrane as in a ribonuclease protection assay is the first step that differs

from a typical 32P-based assay Electroblotting (instead of passive transfer)

from the polyacrylamide gel onto the membrane is the best approach After the

transfer the biotinylated nucleic acids are cross linked to the membrane

B Buffer and Equipment Preparation The BioDetect protocol has been optimized for a 100 cm2 membrane Smaller

or larger membranes can be used with appropriate (proportional) adjustments

to solution volumes

The containers should be clean and RNase-free We recommend treating the

containers with Ambions RNaseZap (Cat 9780) a strong RNase denaturing

solution followed by rinsing with high quality RNase-free water

It is important that the Wash and Blocking Buffers are in solution for use If any

precipitate is visible resolubilize it by warming to 37-6SC Do not however

heat the Streptavidin-Alkaline Phosphatase or the CDP-Star

Bright Star TM Bio detect TM

Use good sterile technique when handling the Wash and Blocking Buffers

Bacterial contamination will cause high background

Dilute the needed amount of 5X wash buffer and 10X assay buffer to 1X with

ddw before use Prepare 5ml of 1X wash buffer per cm2 of membrane or 500ml

of wash buffer for 100cm2 membrane Prepare 2ml of 1X assay buffer per cm2

of membrane or 200ml of assay buffer for 100cm2 membrane

Discard solutions after each incubation The solutions are not reusable

C Detection Procedure


IMPORTANT All incubations described below should be done at room temperature with

constant gentle agitation in containers that allow free movement of the membrane

(eg in flat trays) Also do not allow the membrane to dry Once begun the

membrane must remain wet throughout the procedure

I Wash membrane 2 x 5 min in I X Wash Buffer Do two 5 minute washes in approximately 1 ml Wash Buffercm2

membranewash The Wash Buffer IS provided at 5X so it must be diluted prior to use Generally dilution in ddH2O is sufficient (DEPC treatment andor autoclaving of the water is not necessary)

The end of this washing period is good stopping point if there is not enough time to complete the detection procedure At this point the membrane can be stored wet wrapped in plastic for several days at 40 C

2 Incubate membrane 2 x 5 min in Blocking Buffer Do two 5 minute incubations in approximately 05 ml Blocking Buffercm2

membraneincubation Do not dilute the Blocking Buffer as it is provided as a ready-to-use solution As

mentioned above make sure to resolubilize any precipitate prior to use 3 Incubate membrane 30 min in Blocking Buffer

For this incubation use approximately 1 ml Blocking Buffercm2 membrane

4 Incubate membrane 30 min in diluted Strep-AP Prepare the diluted Strep-Alkaline Phosphatase (Strep-AP) gently and

thoroughly mixing together 10 ml Blocking Buffer and 1μl Strep-AP per typical

size membrane (100 cm2)

Prepare only the volume needed for the experiment at hand because Strep-AP

is not stable for storage when it is diluted in Blocking Buffer

It is important to mix the Strep-AP ith the Blocking Buffer before adding it to

the membrane because if the Strep-AP is added directly to the container with

the membrane and Blocking Buffer heavy and uneven background may result

NOTE Vigorous agitation of enzymes can lead to partial denaturation

5 Incubate membrane 10 min in Blocking buffer Use 05 ml Blocking Bufferjcm2 membrane

(optional) These washes can be extended to 15 minutes each to further

decrease nonspecific background

6 Wash membrane 3 x 5 min in I X Wash Buffer Do three 5 minute washes in approximately 1 ml Wash Buffer cm2

membranewash




7 Incubate membrane 2 x 2 min in IX Assay Do two 2 minute incubations in approximately 05 ml 1lX Assay Buffercm2

membraneincubation

The Assay Buffer is provided at 10X so it must be diluted in high quality water

before use

8 Incubate membrane 5 min in CDP-Star Use very little CDP-Star only about 5 ml100 cm2 The CDP-Star is provided as

a ready-to-use solution do not dilute it

9 Shake off excess CDP-Star and expose membrane to film Quickly blot the membrane on a piece of filter paper to remove excess CDP-

Star without letting the membrane dry If any free-floating CDP-Star is left on the membrane it may generate blotchy background Wrap in a single layer of plastic and expose to film at room temperature

10 About the light emission from CDP-Star CDP-Star reaches peak light emission in 2-4 hours then emission falls to a

plateau which persists for several days Try initial exposures of 1-2 hours

followed by shorter exposures (five min to two hours) the following day if

necessary to obtain the desired image Observation

Fig 1 amp 2 Restriction-digestion of genomic DNA WITH Hind III Xba I Pst I Sac I and

Not I and southern hybridization using 32 Y-ATP labeled probe


MODULE-14

RNA ISOLATION AND RT PCR Object Detection of transcriptional gene expression in genetically modified plants

Principle RT PCR or Reverse transcriptase PCR is another technique by which

gene expression can be studied In order to know if a target gene is expressing or

not RNA is isolated from the tissue mRNA is isolated from total RNA and then it is

converted in to cDNA by Reverse transcriptase The cDNA is amplified by using

gene specific primers Amplification means gene is expressing in the tissue

Requirements Plant material QIAGEN One Step RT ndash PCR kit Solutions Guanidinium thiocyanate buffer 4 M Guanidinium thiocyanate 25 mM Tris-sodium citrate 05 sarcosyl 00007 M β-mercaptoethanol MOPS buffer 01 M MOPS 40 mM sodium acetate 5 mM EDTA pH 70 lodine dye 50 glycerol 025 bromophenol blue 25 Xylene cynol 1mM EDTA) Sample buffer 10mM Tris pH 74 1mM EDTA 30M NaCl Procedure Total RNA was isolated from different stages of the developing wheat spike using Guanidinium thiocyanate protocol developed by Chomczynski and Sacchi (1987) A Isolation of Total RNA 1 Weigh one gram of tissue and immediately transfer it in liquid N2 and ground

to fine powder using pestle and mortar 2 The powder is transferred to 50 ml tubes and 10 ml Guanidinium thiocyanate

buffer is added 3 The tube is gently mixed and 1ml 2 M sodium acetate pH 40 is added

followed by 10 ml phenol ( DEPC treated water saturated ) 2 ml chloroform-


isoamyl alcohol (49 1) mixture Tube with all solutions is mixed gently and placed on ice for 15 min

4 After centrifugation at 10000 rpm for 20 min at 40 C aqueous phase was collected in a fresh tube and an equal volume of isopropanol is added to it The tube is kept at -200 C for 2 hrs

5 After incubation the tube is centrifuged at 10000 rpm for 20 min at 40 C The RNA pellet was washed twice with 3 M sodium acetate pH 55 and then with 70 ethanol

6 Finally pellet was dissolved in 200 microl of DEPC treated water and stored at -200 C

B Electrophoresis of RNA RNA is electrophoresed in denatured agarose gel

1 All glass wares electrophoresis tank comb and casting trays are thoroughly washed with DEPC teated water

2 15 agarose gel is prepared in MOPS buffer 20 microl RNA sample (prepared as 5 microl RNA was mixed with 2 microl 5x MOPS buffer 3 microl formaldehyde and 8 microl of formamide ) is incubated at 650 C for 5 min and cooled on ice

3 2 microl of gel loding dye (50 glycerol 025 bromophenol blue 025 Xylene cyanol and 1mM EDTA) was mixed and loaded on denatured agarose gel

4 Electrophoresis was done and gel was stained with ethidium bromide (05 microl ml) and visualized on UV light

C Purification of mRNA From total RNA mRNA is purified by using oligo dT column supplied with mRNA purification system of the Amersham pharmacia biotech (UK) Kit is based on affinity chromatography and contain oligo- dT cellulose spun column Spun column is inverted several times to resuspend the oligo dT cellulose Top closure was removed followed by bottom closure Column was prepared and used as follows 1 Column is placed upright in a 15 ml centrifuge tube and allowed the storage

buffer to drain out through the column 2 Column is washed twice by adding 1ml high salt buffer (10 mM Tris pH 74 1

mM EDTA 05M NaCl ) and allowing it to drain out under gravity All flow through collected in centrifuge tube are discarded and column is placed in a fresh centrifuge tube

3 RNA sample (dilute up to 1ml in TE buffer) is incubated for 5 min at 650 C 4 Sample is placed on ice and 02 ml sample buffer (10mM Tris pH 74 1mM

EDTA 30M NaCl) is added and mixed gently Sample is applied to the top of cellulose bed in the column (equilibrated) and allowed it to oak under gravity

5 Column is centrifuged at 350g for 2min column is washed twice by adding 025ml high salt buffer (and centrifuging at 350 g for 2min) followed by washing thrice with 025ml low salt buffer (10mM Tris pH 74 1mM EDTA 01M NaCl)


6 All flow through buffer was discarded A 15ml sterile microcentrifuge tube was placed inside 15ml centrifuge tube to collect subsequent effluents from the column

7 Column is placed in 15ml centrifuge tube with its lower end inside the microcentrifuge tube 025ml elution buffer (10mM Tris pH 74 1mM EDTA) pre-warmed at 650 C is added to the column four times (successive) to elute bound poly (A)+ RNA

8 Tube is centrifuged at 350 g for 2min after each application of elution buffer Entire elute was collected in the same sterile tube placed inside the centrifuge tube

9 Spun column is removed from centrifuge tube and microcentrifuge tube is recovered by using clean flamed sterile forceps

10 Eluted sample is transferred aseptically in a fresh bigger centrifuge tube and in the eluted sample 100microl of sample buffer and 10microl of glycogen solution (10mgml) is added followed by 25 ml ice cold ethanol

11 The contents are mixed very gently and the tube is placed at -200 C for over night Precipitated RNA is recovered by centrifugation at 40 C for 10mins

12 Recovered RNA is dissolved in DEPC treated water and stored immediately at - 800 C until next experiment

D Quantitation of m RNA UV spectrophotometer is used for the quantitation of mRNA OD is taken at 260nm as described earlier in DNA quantitation Concentration of mRNA is found out by the following equation Concentration of RNA (microgml) = OD260 x 40 x dilution factor E RT PCR for isolation of gene For RT ndashPCR QIAGEN One Step RT-PCR kit The QIAGEN one step RT-PCR enzyme mix contains a specifically formulated enzyme blend for both reverse transcription and PCR amplification

Omniscript and Sensiscript Reverse Transcriptase are included in the QIAGEN one step RT-PCR enzyme mix and provoid highly efficient and specific reverse transcription Both reverse transcriptases exhibit a higher affinity for RNA facilitating transcription through secondary structures that inhibit other reverse transcriptases Omniscript Reverse Transcriptase is specifically designed for reverse transcription of RNA amounts grater than 50ng and Sensiscript Reverse Transcriptase is optimized for use with very small amounts of RNA (lsaquo50mg) This special enzyme combination provides highly efficient and sensitive reverse transcription of any RNA quantity from 1pg to 2microg

Hot start Taq DNA polymerase included in the QIAGEN One step RT-PCR Enzyme Mix provides hot-start PCR for highly specific amplification During reverse transcription Hot Star Taq DNA Polymerase is completely inactive and does not interfere with reverse transcription reaction After reverse

transcription reactions are heated at 950 C for 15 min to activate Hot Star Taq DNA Polymerase and to simultaneously inactivate the reverse transcriptases This hot-star procedure using Hot Star Taq DNA polymerase eliminates extension from nonspecifically annealed primers and primer- dimers in the first cycle ensuring highly specific and reproducible PCR Although all enzymes are present reaction mix the use of Hot Star Taq DNA Polymerase ensures the temporal separation of reverse transcription and PCR allowing both processes to be performed sequentially in a single tube

1 Template RNA primer solutions dNTP Mix 5x QIAGEN One Step RT-PCR Buffer and RNase-free water thawed and placed on ice

2 Master mix is prepared as follows 1x QIAGEN OneStep RT-PCR Buffer 400 microl of each dNTP 100 ng of each primer 2 microl QIAGEN OneStep RT-PCR Enzyme Mix 200 ng of template RNA RNase- free water to make volume to 50 microl

3 The master mix is mixed thoroughly and dispended approximately to the individual PCR tubes Then mixed gently by pipetting it up and down few times

4 Template RNA (200 ng) is added to individual PCR tubes 5 Thermal cycler is programmed as follows

Reverse transcription at 500 C for 30 minutes PCR activation for 15 min at 950 C Subsequent 35 cycle of 940 C denaturation for 30 Sec 550 C annealing for 30 sec 2 min extension at 720 C Final extension is done at 720 C for 10 min

6 RT ndash PCR program is started PCR tubes were still on ice When thermal cycler reached 500C PCR tubes were placed in the thermal cycler

F Analysis of amplification products

After the completion of PCR cycles 10 microl of the amplicon is analyzed on 2 agarose gel by electrophoresis and results were recorded in gel documentation system Observation


RT-PCR for transcriptional gene expression of gene using RNA isolated from different wheat plants


MODULE-15

WESTERN BLOTTING Object Western blotting for confirmation of transgene expression in terms of

protein of desired molecular weight

Principle Western blotting - also known as protein blotting or immunoblotting- is a well-established method used to detect a target protein from a mixture of proteins This method can determine the expression levels of the target protein in selected cells or tissues either under normal or experimental conditions The Western blotting protocol is made of five steps 1 Electrophoresis 2 Blotting 3 Labeling 4 Detection 5 Image AnalysisProtein Electrophoresis and Blotting Proteins in a mixture (eg cell culture lysate tissue homogenate etc) are separated by molecular weight or isoelectric point using polyacrylamide gel electrophoresis (PAGE) The proteins are transferred (blotted) from the gel to a membrane (nitrocellulose or PVDF) for easier handling and manipulation Labeling Following the blotting step the target protein is labeled using antibodies The primary antibody which is specific for the target protein can be labeled or unlabeled To maximize sensitivity and signal-to-noise ratio most Western blotting procedures use an unlabeled primary antibody and a conjugated or labeled secondary antibody (the secondary antibody is specific for the primary antibody) The secondary antibody can be radiolabeled labeled with a dye or other molecule or conjugated with an enzyme Typical enzymes used are horse radish peroxidase (HRP) and alkaline phosphatase (AP) both of which use a detection reagent to generate a signal that can be quantitated The signal can be the production of an insoluble dye (chromogenic) or the generation of light (chemiluminescent or chemifluorescent) Detection and Image Analysis Target proteins are detected using the appropriate detection reagents to generate a signal that can be quantitated For chromogenic methods the signal is captured directly on the membrane For radiolabeling chemiluminescent and chemifluorescent methods the signal is captured using film or an imaging system The acquired image is quantitated using image analysis software


(A) TRANSFER OF PROTEINS FROM GEL TO NITROCELLULOSE MEMBRANE

Requirements Reagents Transfer buffer 1 litre

Tris Base (156mM) 193g Glycine (120mM) 90g

Add double distilled water to make 1 litre pH should be between 81 amp 84 without adjustment Can also be made up as a 20X stock solution

Procedure

1 Cut the Nitrocellulose membrane and whatman 3 microm filter paper of the size

of the gel

2 Mark the gel and membrane for proper orientation

3 Soak NC membrane in transfer buffer for 15-20 min

4 Soak the gel also for 5 min

5 Arrange the sandwich as follows in the following order

Black panel

Well soaked fiber pad

One wetted sheet of Whatman paper (3microm)

Carefully place the gel on wet filter paper (avoid air bubbles)

Wet the gel with transfer buffer

Carefully lay a wetted sheet of NC beginning from one side to avoid air

bubbles

Place a wetted sheet of 3 microm paper over NC

Cover with second soaked fiber pad

Close the transfer cassette

6 Slide it into the buffer tank (Black panel of cassette on the same side as the

black panel of electrode assembly)

7 Fill the tank with Transfer buffer

8 Attach to electrodes and switch on the assembly at 100V for 1 hour or 30V

for overnight transfer

(B) DETECTION OF PROTEIN BY ANTIBODIES Requirements Solutions to prepare


1 TBS (50mM Tris ndashHCl pH - 80 1500 mM NaCl)

2 3 BSA in TBS or 1 SMP in TBS

3 05 BSA

Membrane Blocking 1 Remove membrane from transfer apparatus to a small container (Petri

plates)

2 Add at least 8 ml 3 BSA TBS (enough to cover the membrane)

3 Rock gently for 30min to 1 hour (Membrane can be stored in this solution

with 1mm NaN3 overnight)

4 Washing Pour off BSA solution amp rinse briefly with TBS three times

5 Primary Antibodies wash (110 to 1100000)

Pour off TBS add 1st Ab at appropriate dilution in 05 BSA TBS

Rock gently for at least 1hr

Overnight incubation is possible amp may induce detection sensitivity

6 Washing Pour off 1st Ab solution

Wash twice for 10min with TBS

7 Secondary antibody wash Pore off TBS

Add secondary Ab at appropriate dilution in 05 BSA

Rock the membrane gently for at least 1 hr

Overnight incubation can also be done

8 Washing Pour off secondary Ab solution and rinse for 30 min with TBS with 3 change


1 Alkaline phosphatase buffer Tris-HCl (pH 95) 01M

NaCl 01M

MgCl2 5mM

2 BCIP in 100 DMF 50 mgml 3 P-NBT in 70 DMF 50 mgml

Developing NC membrane Alkaline Phosphatase

AP-Ab react with a BCIP-NBT (5-Bromo 4-chloro Inolylphosphate - NitroBlue

tetrazolium) substrate to give a dark purple precipitate

Product is stable amp will not fade

Wash NC for 5 min in ABP

Prepare developing reagent (Use within 1hr) Take 66microl NBT then add 10 ml

AP Band mix well After that add 33microl BCIP solution

Pour off APB amp add 10 ml developing reagent

Incubate at room temperature at 37˚C for 30 min

Can be done overnight to increase signal

Stop reaction by rinsing the membrane with 20mM EDTA in TBS

Observation

Western Blotting


FOREWORD

CBSH GBPUAampT Pantnagar INDEX

MODULE-1

MODULE-2

MODULE-3

QUANTITATIVE ANALYSIS OF PLANT GENOMIC DNA


MODULE-4

AGAROSE GEL ELECTROPHORESIS

MODULE-5

GEL DOCUMENTATION SYSTEM

MODULE-6

PCR AMPLIFICATION

MODULE-11

Development of immuno-dipstick test for detection of transgene expression in genetically modified plants

MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14

RNA ISOLATION AND RT PCR

MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3


Object Quantitation of genomic DNA isolated from leaves and seeds of transgenic plants


Principle Agarose is extracted from a see weed and is a linear polymer of different forms of galactose Agarose gels can be casted by melting the agarose in the presence of desired buffer until a clear transparent solution is obtained The melted solution is then poured into a mold and allowed to harden Upon hardening the agarose forms a matrix the density of which is determined by the concentration of the agarose When an electric field is applied across the gel DNA which is negatively charged at natural pH migrates towards the anode

Equipments required Horizontal slab gel electrophoresis apparatus procured from Bangalore genei

Solutions

Procedure

A Gel preparation

B Loading and running the gel

C Visualization and photography of DNA in agarose gel

MODULE-5


MODULE-6

PCR AMPLIFICATION

Procedure Dot blot is an efficient technique for detection of proteins for it is very rapid technique and also semi quantitative in nature

MODULE-11

Object Development of immunodipstick test for detection of Bt transgene expression in genetically modified plants

Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING

Solutions Reaction Mixtures

PROTOCOL

Problems and Remedies

Problem 1 Endonuclease or exonuclease contamination

Blot hybridizations (eg Northern or Southern blot)

B Buffer and Equipment Preparation

MODULE-15

Object Western blotting for confirmation of transgene expression in terms of protein of desired molecular weight

Membrane Blocking

Requirements

Reagents


PREFACE

Transgenic crops have become reality as then offer great opportunity to move gene(s) across the sexual barrier in more precise and faster ways as well as introgression of traits that would reduce the cost positive environmental impact and enhance the nutritional value Transgenic crops are not a panacea for solving all the problems however there are a number of perceived ecological and economic threats that need to be addressed while producing and deploying transgenic crops for food and nutritional security Considering these threats the training workshop is organized to cater the needs of scientific personals who wish to acquaint themselves about Biosafety related issues in monitoring and evaluation of transgenic crops under field conditions and its impact on biodiversity The main goal of this training is to scintillate the mind of the Scientific Community for realizing the scope and limitations of transgenic technology for sustainable agricultural growth in the country It will cover both relevant lectures and practical concepts of molecular testing methods for evaluation of transgenic crops The training workshop strives to provide a packed module of exercises in systematic manner for on- hands training The faculty members of Department of Molecular Biology and Genetic Engineering namely Dr Sonu Ambwani Dr BR Singh Dr Sneh Narwal Dr Dinesh Pandey Dr Gohar Taj Khan and Research Staffrsquos Mahender Singh Dr Kaushal Gautam who worked round the clock to organize the training workshop and prepare the manual to make it useful to the participants The contributions of non-teaching staffs and contractual workers as Mr AVS Rao Mr RN Singh Mr Nathoo Parsad Mr Abhilakh Singh are gratefully acknowledged Further training workshop will not be possible without visionary leadership of Prof GK Garg Advisor and Coordinator Biotechnology as well as Course Director of present training The painstaking efforts of our guest internal faculty is duly acknowledged The training workshop has been organized under world bank-GEF project and sponsored by Ministry of Environment and Forests The immense interests shown by Department of Biotechnology Govt of India for establishing this National Facility on Biosafety at Pantnagar is duly acknowledged

Dr Anil Kumar Course Organizer Associate Professor Dept of Mol Biol amp Genetic Engg

CBSH GBPUAampT Pantnagar


INDEX

MODULE-1


MODULE-2







MODULE-5


MODULE-6


MODULE-7


MODULE-8


MODULE-9


MODULE-10



MODULE-11


MODULE-12


MODULE-13







Contents MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45


MODULE-1


plants









40C



14 M NaCl 20475 g


02 BME 50 μl






and mortar




swirling


emulsify










Observation

M A




MODULE-2


transgenic plants





05 M EDTA 10 ml

SDS 40 g

NaCl 03 g

H2O 65 ml




H2O 30 ml


temperature












40C






shaken




for 15 minutes




Observation


Where M= Marker

A= DNA

M A



MODULE-3


plants


















OD at 260nm

OD at 280nm

Ratio OD260OD280

Yield (microg)



MODULE-4














Solutions




05M EDTA (pH 80) 100 ml




Boric acid 55



at RT

















buffer


with micro-pipette



system








Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


INDEX

MODULE-1


MODULE-2







MODULE-5


MODULE-6


MODULE-7


MODULE-8


MODULE-9


MODULE-10



MODULE-11


MODULE-12


MODULE-13







Contents MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45


MODULE-1


plants









40C



14 M NaCl 20475 g


02 BME 50 μl






and mortar




swirling


emulsify










Observation

M A




MODULE-2


transgenic plants





05 M EDTA 10 ml

SDS 40 g

NaCl 03 g

H2O 65 ml




H2O 30 ml


temperature












40C






shaken




for 15 minutes




Observation


Where M= Marker

A= DNA

M A



MODULE-3


plants


















OD at 260nm

OD at 280nm

Ratio OD260OD280

Yield (microg)



MODULE-4














Solutions




05M EDTA (pH 80) 100 ml




Boric acid 55



at RT

















buffer


with micro-pipette



system








Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


MODULE-11


MODULE-12


MODULE-13







Contents MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45


MODULE-1


plants









40C



14 M NaCl 20475 g


02 BME 50 μl






and mortar




swirling


emulsify










Observation

M A




MODULE-2


transgenic plants





05 M EDTA 10 ml

SDS 40 g

NaCl 03 g

H2O 65 ml




H2O 30 ml


temperature












40C






shaken




for 15 minutes




Observation


Where M= Marker

A= DNA

M A



MODULE-3


plants


















OD at 260nm

OD at 280nm

Ratio OD260OD280

Yield (microg)



MODULE-4














Solutions




05M EDTA (pH 80) 100 ml




Boric acid 55



at RT

















buffer


with micro-pipette



system








Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


Contents MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45


MODULE-1


plants









40C



14 M NaCl 20475 g


02 BME 50 μl






and mortar




swirling


emulsify










Observation

M A




MODULE-2


transgenic plants





05 M EDTA 10 ml

SDS 40 g

NaCl 03 g

H2O 65 ml




H2O 30 ml


temperature












40C






shaken




for 15 minutes




Observation


Where M= Marker

A= DNA

M A



MODULE-3


plants


















OD at 260nm

OD at 280nm

Ratio OD260OD280

Yield (microg)



MODULE-4














Solutions




05M EDTA (pH 80) 100 ml




Boric acid 55



at RT

















buffer


with micro-pipette



system








Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


MODULE-1


plants









40C



14 M NaCl 20475 g


02 BME 50 μl






and mortar




swirling


emulsify










Observation

M A




MODULE-2


transgenic plants





05 M EDTA 10 ml

SDS 40 g

NaCl 03 g

H2O 65 ml




H2O 30 ml


temperature












40C






shaken




for 15 minutes




Observation


Where M= Marker

A= DNA

M A



MODULE-3


plants


















OD at 260nm

OD at 280nm

Ratio OD260OD280

Yield (microg)



MODULE-4














Solutions




05M EDTA (pH 80) 100 ml




Boric acid 55



at RT

















buffer


with micro-pipette



system








Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents



and mortar




swirling


emulsify










Observation

M A




MODULE-2


transgenic plants





05 M EDTA 10 ml

SDS 40 g

NaCl 03 g

H2O 65 ml




H2O 30 ml


temperature












40C






shaken




for 15 minutes




Observation


Where M= Marker

A= DNA

M A



MODULE-3


plants


















OD at 260nm

OD at 280nm

Ratio OD260OD280

Yield (microg)



MODULE-4














Solutions




05M EDTA (pH 80) 100 ml




Boric acid 55



at RT

















buffer


with micro-pipette



system








Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


MODULE-2


transgenic plants





05 M EDTA 10 ml

SDS 40 g

NaCl 03 g

H2O 65 ml




H2O 30 ml


temperature












40C






shaken




for 15 minutes




Observation


Where M= Marker

A= DNA

M A



MODULE-3


plants


















OD at 260nm

OD at 280nm

Ratio OD260OD280

Yield (microg)



MODULE-4














Solutions




05M EDTA (pH 80) 100 ml




Boric acid 55



at RT

















buffer


with micro-pipette



system








Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents



40C






shaken




for 15 minutes




Observation


Where M= Marker

A= DNA

M A



MODULE-3


plants


















OD at 260nm

OD at 280nm

Ratio OD260OD280

Yield (microg)



MODULE-4














Solutions




05M EDTA (pH 80) 100 ml




Boric acid 55



at RT

















buffer


with micro-pipette



system








Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents

MODULE-3


plants


















OD at 260nm

OD at 280nm

Ratio OD260OD280

Yield (microg)



MODULE-4














Solutions




05M EDTA (pH 80) 100 ml




Boric acid 55



at RT

















buffer


with micro-pipette



system








Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


MODULE-4














Solutions




05M EDTA (pH 80) 100 ml




Boric acid 55



at RT

















buffer


with micro-pipette



system








Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents














buffer


with micro-pipette



system








Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents





Stain _________________________________________



1 2 3 4 5 6 7 8 9 10


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


MODULE-5









publishing








Desktop









sample type
























is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


















is should be saved

















MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents




MODULE-6














no of cycles




reaction mixture

2 Template DNA















fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents



fidelity






products












amplification












4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents





4 Assay buffer




5 DNA template



6 Control primers









11 PCR tubes


13 50X TAE 40ml



dNTP mix 3 μl






















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents















fragment

Observation





MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


MODULE-7













for 20 min









MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents

MODULE-8



PRINCIPLE



























Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents



Total Volume (μl)










graph


MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents

MODULE-9










methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents



methanol










Glycine 192 mM

SDS 01





01 CBB - R250






10 SDS 01ml

10 APS 02ml


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


TEMED 10μl

ddw 669ml



10 SDS 80μl

10 APS 80μl

TEMED 10μl

ddw 453ml






















the gel


dye solution




Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents



Observation

M A1 A2

(SDS-PAGE)

Where

M= Marker

A1 = Non Bt Protein

A2= Bt Protein




MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


MODULE-10





































substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents





substrate

PBS (1L)

Observation




MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


MODULE-11

















provided









Bt- cotton sample



PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents

PRECAUTIONS




activity











considered positive

Observation




MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


MODULE-12










(DGH030)



by sandwich ELISA

Materials provided





10x buffer A


Stop solution

Solutions







substrate


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


Sample preparation









Procedure






buffer








Observation



MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


MODULE-13





























DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents



DNA - 5 μg

10x buffer - 50μl





















to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents







to set for 20min











salt










polyacrylamide gels








down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents






down
















incomplete transfer















relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents







relative to the gel













experiments






















































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents









































dH2O and air dry










denaturation









at 7000 xg
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents
























should be lt2 cm






tube











labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents






labeled probe



















use





















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


















to solution volumes









































membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


























membranewash





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents





membraneincubation


before use












MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents


MODULE-14


















































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents








































MODULE-15









Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents






Procedure


of the gel





Black panel






bubbles













3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents




3 05 BSA


plates)


















NaCl 01M

MgCl2 5mM













Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents








Observation

Western Blotting


FOREWORD


MODULE-1

MODULE-2

MODULE-3



MODULE-4


MODULE-5


MODULE-6

PCR AMPLIFICATION

MODULE-11


MODULE-12

MODULE-13

SOUTHERN BLOTTING

MODULE-14


MODULE-15


MODULE-1 4

MODULE-2 6

MODULE-3 8

MODULE-4 9

MODULE-5 11

MODULE-6 13

MODULE-7 17

MODULE-8 18

MODULE-9 19

MODULE-10 22

MODULE-11 23

MODULE-12 25

MODULE-13 27

MODULE-14 40

MODULE-15 45

MODULE-1

Procedure

MODULE-2

MODULE-3






Solutions

Procedure

A Gel preparation



MODULE-5


MODULE-6

PCR AMPLIFICATION


MODULE-11


Materials

Procedure

MODULE-12


Sample preparation

Procedure

MODULE-13

SOUTHERN BLOTTING


PROTOCOL





MODULE-15


Membrane Blocking

Requirements

Reagents

development of dot immunobinding assay … training has been organized which will include both...

Documents