DNA FINGERPRINTING

MATERIALS & METHODS

.

SAMPLE COLLECTION

6 verities of chili plants were collected from G.KV.K agri university, Bangalore. The samples are stored at 4⁰c.

Guntur

Sarca aroka

Samruthi

Indm kaddi

Capsium-L

SBL-C

DNA EXTRATION

(CTAB Method)

REQUIREMENTS: CTAB extraction buffer 0.3M Sodium Acetate. Phenol : Chloroform : Isoamyl Alcohol Isopropanol Ribonuclease 70% Ethanol 5M Sodium Chloride TE buffer Chloroform : Isoamyl Alcohol

DAY 10.15g - 0.3g in 700- 900μl of CTAB buffer

200 each time & crush with the help of Motor & pestel

Transfer to 2ml of vials & incubate T 50°C for 15mins in water bath

After incubation add equal vol of chloroform: isoamyl alcohol (24:1)

Incubate at 37°C for 30mins in shaker

Centrifuge at 12000rpm for 12mins at R.T

Collect the upper layer & transfer t 2ml vials.

Add 0.5v 5m Nacl & mix it well & then add full vol of isopropanol

Storage for -20°C overnight

DAY 2Centrifuge at 12000rpm for 12mins at

4 C

Collect pellet & allow to air dry for 10mins

After air dry add 800of TE buffer

Add 6 of Rnase & incubate at 37 C for 30mins in the bath

After incubation add equal vol of phenol: chloroform: isoamly alcohol (25:24:1)

Centrifuge at 12000rpm for 12mins at R.T

After centrifuge we get three layer

Collect the upper layer & transfer to 2ml vials

Add equal vol of chloroform : isoamyl alcohol & mix it gently

Centrifuge at 12000rpm for 12mins at R.T

Collect upper layer & transfer to 2ml vials

Add 0.1v 3m sodium acetate & mix it well

Add full vol of absolute ethanol & mix it well

Store at -20°C overnight

DAY 3

Mix & centrifuge at 12000rpm for 12mins at 4°C

Collect the pellet & then add 1.5ml 70% ethanol

Dissolve pellet gently

Centrifuge at 12000rpm for 12mins at 4°C

Collect pellet & air dry for 10 – 15 mins

Add TE buffer

Dissolve the pellet gently

Prepare 0.8% of agarose gel

Load the 10μl genomic DNA

AGAROSE GEL ELECTROPHORESIS

REQUIREMENTS :

Electrophoretic unit

Methanol

Electrophoretic medium

Solid support (1.2%agarose)

Gel loading buffer

Gel-sealing tape

Micropipette with disposable tips

Microfuge vials

Sample

Uv Transilluminator

Stain

Seal the edges of clean casting tray

Prep sufficient buffer (1x TAE ) to fill electrophoresis tank to cast the gel

Prepare the soln of agarose in electrophoresis buffer at 0.8% conc

When molten has cooled add ETBR

Choose appropriate comb for forming the slots in gel & pour soln

Allow the gel to set completely, remove the comb & tape carefully

Mount the gel in the electrophoresis tank

Then pour the electrophoresis buffer to cover the gel to a depth of 1mm.

Slowly load the sample mixture into the slots using disposable micropipette at 50-100v

sample or dye is migrated a sufficient distance through the gel, turn off the electric current

Examine the gel by UV light and photograph the gel

SPECTROPHOTOMETRIC QUANTIFICATION OF DNA

REQUIREMENTS :

UV spectrophotometer

TE buffer

DNA sample

Micropipette

Absolute Ethanol

PROCEDURE:

Prepare a known dilution of DNA sample in the TE buffer, which is used to dissolve the DNA sample.

Calibrate the spectrophotometer for blank using TE buffer.

Record the OD of the sample at 260nm and 280nm.

Calculate the concentration of DNA in the sample using the Relation

QUALITY PCR

REQUIREMENTS :

Thermo stable Taq DNA polymerase

dNTP mix (10mM)

Chili genomic DNA

Sterile distilled water

PCR buffer (10x)

Forward primers and reverse primer specific to positive control

Micropipettes of different ranges

S

.

N

o

DNA sample

volume(µl)

Positive

control (µl)

dNTPs (µl) Buffer (10x)

(µl)

Forward

primer (µl)

Reverse

primer (µl)

Taq

polymerase

(µl)

Sterile

water (µl)

1 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

2 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

3 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

4 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

5 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

6 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

Reaction components

QUALITY PCR PROGRAMHeated lid 110ºC

Pre- heated lid off

Pause- off

Initial denaturation- off

Loop 1 (initial denaturation)

No. of cycles 1

Segment 94ºC 3minutes

Loop 2

No. of cycles 30

Segment 94ºC 30sec

Segment - 55ºC - 30sec

Segment - 72ºC - 1minute

Final extention 72ºC - 5minutes

Final hold 10ºC

AMPLIFICATION OF DNA USING RAPDREQUIREMENTS :

Thermostable Taq DNA polymerase

dNTP mix (10 mM)

Template DNA

Sterile distilled water

PCR buffer (10x)

Oligonucleotide primers

Ice bucket

Eppendorff vials

Micropipettes of different ranges

Thermal cycle

Ingredients Volume to be taken

Template DNA 10.0μl

dNTPs 2.5μl

PCR buffer 2.5μl

Primers 1.0μl

Taq DNA polymerase 0.75μl

Sterile water 8.25μl

Total 25μl

PROCEDURE:

Set up the following reaction mixture (25 l) in the same order.

All those mentioned ingredients are mixed and prepared for the total no of reactns including a blank with a particular primer excluding template

The calculated volume of masters mix ix then transferred to labeled PCR tubes with template source and primer.

Finally 0.33µl of Taq DNA polymerase is added to each tube

The contents of the tube are mixed with a brief spin and transferred to PTC 200 thermal cycler

The program with following conditions is selected for the amplification

Number cycles 30

Segment 94.0ºC 1minute

Segment 35.0ºC 1minute

Segment 72.0ºC 1minute

UREA POLYACRYLAMIDE GEL

ELECTROPHORESISREQUIREMENTS :

Vertical electrophoresis unit

Urea 7M

Acrylamide 40%

10x TBE (Tris Borate EDTA) buffer

10%Ammonium Per Sulphate (APS)

Tetra Ethyl Methylene Diamine (TEMED)

Gel loading dye

Autoclaved distilled water

PROCEDURE :

Preparation of gel (50ml)

Weigh 9.08g of urea and dissolved by heating in about 15ml

autoclaved distilled water.

Add 6.25ml of 40% acrylamide and 5ml of 10x TBE buffer.

Make up the volume to 50ml with autoclaved distilled water.

Add 350μl of APS and 35l of TEMED and mix well.

Immediately transfer the gel into the previously arranged vertical electrophoresis unit.

Electrophoresis of the DNAPre-run the gel for about one hour at 100V.

To the PCR sample add 4.2l of gel loading dye.(Xylene Cyanol).

Boil the samples for 10minutes at 85-90C.

Immediately chill the sample in ice for 2minutes.

Spin the sample at 3000rpm for 2minutes and load in top the gel.

The electrophoresis is carried out at 150V tll the dye front reaches the bottom of the base plate, the plates are cooled with an ice pack during the run to prevent over-heating.

SILVER STAINING

REQUIREMENTS :

Gel container

Shaker incubator

10% acetic acid

de-ionised water

autoclaved double distilled water

silver nitrate solution

2.5% sodium carbonate and 0.02% formaldehyde.

PROCEDURE :

Place the gel in 5 volumes of a mixture of 30% ethanol and 10% acetic acid.

Incubate the gel for 3 hours or overnight with shaking at room temperatures.

Remove the ethanol / acetic acid solution and add 5 gel volume of 30% ethanol.

Incubate for 30minutes at room temperatures with shaking. Repeat this step twice.

Remove the ethanol solution and add 10 gel volume of deinonised water.

Incubate for 10minutes at room temperatures with shaking. Repeat this step twice.

Remove the deionised water and add 5 gel volume of 0.1%silver nitrate solution.

Incubate for 30minutes at room temperatures with shaking.

Remove the silver nitrate solution and wash the gel for 20seconds under a stream of deionised water.

Add 5 gel volume of a mixture of 2.5%sodium carbonate and 0.02% formaldehyde.

Incubate at room temperature with shaking. Bands will start appearing slowly.

Incubate until band appears.

Stop the reaction by washing with 1% acetic acid.

Wash several times with deionised water for 10 minutes each

The gel might now be observed over an illuminating source of white light for better result and documented.

For preserving the gel, place it in 20ml of a 20% glycerol solution.

Keep the gel between two layers of gelatin [aper and dry for 3 days at 37ºC.

THANK YOU

-SACHIN SUBBA

DEPT OF BIOTECH

CMRIT, BANGALORE