Rules of occupational safety

1. Only practising students, specified by the timetable, are right of entry at the practical classes. No admittance of any visitors. Authorized personnel only.

2. Students are required to familiarize with their task. Laboratory coats and working instructions are obligatory. Long hair must be adapted for working with a burner without any risk of injury. Overgarments and bags must be put on the given place.

3. Any leaving is allowed just with a lecturer´s permission. 4. Only prescribed activities are allowed in laboratories. No eating, no drinking, no smoking and no storing

food in laboratories. Laboratory equipment is not allowed to use for any other purposes. 5. If there is a leakage of harmful chemicals possible, the extraction must be ensured. Working with fuming

substances, substances irritating to the respiratory, toxic gases and vapours, as well as annealing and combustion is allowed to do just in a fume cupboard.

6. Students must be careful during the manipulation with a safety bulb pipette filler. Pieces of broken glass must be put in a specific container, label "GLASS".

7. It is possible to pour out only the solvents perfectly miscible with water into the sink. They must be sufficiently diluted (at least 1:10), maximum of 0.5 litre. Aqueous solutions of acids and alkalis must be diluted at least 1:30. Solvents immiscible with water, poisons, acids and alkalis over the given concentration and substances loosing toxic gases and gases irritating to the respiratory must be disposed into the special waste container.

8. An acid is pouring into the water during the dilution of acids, never vice versa. 9. It is forbidden to suck in solution into a pipette per mouth. A safety bulb pipette filler must be used. 10. Spilt acids must be washed by water immediately, if need be neutralized by sodium carbonate. Spilt

alkalis must be just washed by water. 11. All burners and electrical current must be switched off due to spilling of flammable liquids and it is

necessary to clear the air. Pouring liquids must be absorbed by suitable porous material and it is liquidate in the appropriate way.

12. During the heating of a liquid in a boiling flask superheating must be prevented by using a boiling chip. 13. It is necessary to check all devices before the start of working. Possible faults and defects must be

reported to a lecturer or a laboratory technician. 14. Intentional handling with electrical device and substances is forbidden. To switch on a device and to light

a burner is allowed by the approval of a lecturer or a laboratory technician. 15. All centrifugation procedures must be controlled by a lecturer or a laboratory technician. Vessels for the

centrifugation must be well balanced and the top of the centrifuge must be closed safely during the operation.

16. The gas intake and electrical current must be switch off and clear the air if there is a leakage of gaseous fuels.

17. A lighted burner without supervision is not allowable. If there are any problems with a turner, it is necessary to switch off the gas intake and the burner must be regulated.

18. Students are obliged to inform a lecturer of any accident, injury, or in case of ingestion chemicals. 19. Serious breach of rules because of a lack of discipline or ignorance is the reason of leaving the practical

classes as an unexcused absence. 20. Students must be informed about classification of toxic, carcinogenic, mutagenic and damaging fertility

substances. Safety sheets of particular substances are available in laboratories. 21. Students must be informed about rules of occupational safety with highly toxic substances (label T+)

using in laboratories (e.g. mercury, potassium cyanide, ethidium bromide, mercury(II) nitrate).

Pilsen, September 16th, 2016 Ing. Václav Babuška, Ph.D.

Deputy Head of the Department

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Pipetting training 1) Pipetting of distilled water with checking of the precision by weighting a) Pipetting of large volumes, pipettes with fixed volume

Place a small empty beaker on a balance pan. The mass of the empty vessel is called the tare. Press the TARE button to get a reading of 0.000 g. Take the beaker from the balance, place it on the table and pipette into it distilled water of the following volumes:

3 x 2000 µl 2 x 500 µl

For pipetting of 2 ml (=2000 µl), use the pipette with the fixed volume. Select the proper tip for the pipette (big white). To attach the tip, firmly press the shaft of the pipette into the large open end of the tip with light force to ensure a good seal. Similarly, for pipetting of 0.5 ml (=500 µl), use the pipette with the fixed volume and proper tip.

After you finish the pipetting, place the beaker on a balance pan to read the weight of distilled water inside. Compare the result with a theoretical expected value obtained as a sum of pipetted volumes recalculated to mass using the density. For doing this, density of distilled water at the temparature in the laboratory is ρ=1.000 g/cm3.

Expected volume Expected mass Measured mass

b) Pipetting of small volumes, pipettes with adjustable volume

Place an empty small plastic test tube of a volume 1.5 ml, so called Eppendorf tube, on a balance pan, and press the TARE button.

Take the open Eppendorf tube in your hand and pipette into it distilled water of the following volumes:

375 µl 25 µl

For pipetting of 375 µl, use the pipette with adjustable volume in the range 100-1000 µl and proper tip. For pipetting of 25 µl, use the pipette with adjustable volume in the range 20-200 µl and proper tip (yellow).

After you finish the pipetting, close the Eppendorf tube and place it on a balance pan to read the weight of distilled water inside. Compare the result with a theoretical expected value obtained as a sum of pipetted volumes recalculated to mass using the density. For doing this, density of distilled water at the temparature in the laboratory is ρ=1.000 g/cm3.

Expected volume Expected mass Measured mass

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2) Determination of the density of an unknown solution Place an Eppendorf tube on a balance pan and press the TARE button.

Into the Eppendorf tube, pipette exactly 1.000 ml (=1000 µl) of a solution, density of which you want to determine.

For pipetting of 1000 µl, use the pipette with adjustable volume in the range 100-1000 µl and proper tip.

Close the Eppendorf tube and place it on a balance pan to read the weight of a solution inside. From a known volume and mass measured, calculate the density.

Volume Mass Density

3) Preparation of a solution and its aliquoting into individual tubes

Pipette into an Eppendorf tube: distilled water 93 µl dye solution 7 µl For pipetting of 93 µl, use the pipette with adjustable volume in the range 20-200 µl and proper tip (yellow). For pipetting of 7 µl, use the pipette with adjustable volume in the range 0.5-10 µl and proper tip (very small white). At this step, you are adding a very small volume. The best way how to do it: The orifice of the tip must be dipped below the level of the solution which is already in the Eppendorf tube. By adding these 7 µl, you will simultaneously mix the solution, read further how to do this!

Mix thoroughly the content of the Eppendorf tube. It can be done by so called "pipetting up and down" several times, i.e. repeatedly pressing and releasing the button of the pipette causing movement of the solution in the tip "up and down". In this exercise, the solution is coloured, so you can see what is happening and check if mixed sufficiently. Taking the tip out of the Eppendorf tube, be careful to make the tip empty! Prepare 5 microtubes (0.2 ml) in a rack and pipette into each exactly 20 µl of the solution you have prepared.

5 x 20 µl 100 µl Evaluate the precision and accuracy of your pipetting!

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Paper chromatography of amino acids

1. Take a sheet of chromatographic paper (18 x 18 cm). Find out a centre of the sheet. Draw

a circle around the centre about 3 cm in diameter. You can use prepared template. This is the "base-line", the start position. On the base-line make 6 marks evenly spaced and number them 1-6.

Chromatogram 2. Using labelled capillaries make small spots (less than 0.5 cm in diameter) of about 5 µl of

the standards and your unknown sample. The application should be repeated 2 times. Before the further application, the wet spots must be dried under the infra-red lamp or a ventilator.

3. Use your pencil to perforate the paper exactly in the centre to draw through the hole

a paper "wick". Place the paper into a Petri dish partly filled with a solvent mixture (n-butanol, acetic acid, water – 4:1:1) and cover the paper with a cap. Do not take the Petri dish out from the fume chamber! All following procedures must be done in a fume chamber!

Petri dish with solvent chromatogram paper wick

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4. The chromatograms are left to develop until the head of a solvent overlaps the Petri dish cap. The paper is removed, dried, and sprayed with ninhydrine reagent. Heat the paper at 80°C in order to develop the formation of blue complexes.

5. Calculate the individual Rf according to the equation.

�� =distancebetweenbaselineandthecentreofthespot

distancebetweenbaselineandthefrontofthesolvent

Site Amino acid Rf

1 Lysine

2 Glycine

3 Alanine

4 Isoleucine

5 The mixture of 1-4 - - -

6 Unknown sample

Sample identification:

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Separation of plant pigments by thin-layer chromatography

There are a few lipophilic pigments in green parts of plants. They have different chemical and functional properties. The primary function of pigments in plants is photosynthesis which uses the green pigment chlorophyll along with several red and yellow pigments. These pigments can be separated using Thin Layer Chromatography (TLC).

Procedure:

1. There are prepared samples of green leaves or needles on the working place. Cut it in pieces in a mortar. Then add a small spoon of calcium carbonate, a little bit of glass shards and crush it with a pestle in the presence of 1-2 ml of acetone. Protect your eyes using glasses.

2. Take two TLC plates. After the separation, one is for you to be attached to the protocol, the second will be used further and destroyed by procedure of extraction of one of the pigments.

3. Draw the base-line about 2 cm from the shorter edge of the plate. Use the thin pencil.

4. Dip the glass capillary into the acetone extract and touch the tip of the capillary to the

plate at the position you’ve marked. Gently and quickly make spot according to the picture. Try to make as small spots as possible and avoid scratching of the thin layer. For higher concentration repeat the process of application for several times.

5. Prepared separation chamber should be kept close. There is approximately 1cm thick layer of liquid solvent mixture on the bottom and the rest volume is filled with its vapors.

6. Place the thin layer plate carefully in the chamber (chromatography tank), containing a solvent at the bottom. Cover the vessel with a glass plate and allow to separate about 15 min. The base – line must be situated above the level of a solvent.

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7. When the front of a solvent almost overlaps the top of the TLC plate, remove the plate. Evaluate the dry plate for the presence of pigments and enclose to laboratory protocol.

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Measurement of absorption spectrum of one of the pigments After separation of the plant pigments, take one of the TLC plates and select one of the green strips. Use the scissors to cut out the strip from the TLC plate. Put the piece with the strip into a test tube and add 2 ml of ethanol. Wait until complete discoloration of TLC plate piece.

Measure absorption spectrum of this fraction using a spectrophotometer in a range of wavelengths from 350 to 700 nm. As a blank solution, use distilled water. By comparing the spectrum with spectra of individual pigments, determine whether the fraction contains chlorophyll a or chlorophyll b.

wavelength nm A

wavelength nm A

wavelength nm A

350 470 590 360 480 600 370 490 610 380 500 620 390 510 630 400 520 640 410 530 650 420 540 660 430 550 670 440 560 680 450 570 690 460 580 700

Name of the pigment

0

0,05

0,1

0,15

0,2

0,25

350 400 450 500 550 600 650 700nm

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Separation of dye mixture by gel chromatography

Task: Separate dextran blue and potassium chromate.

Dextran blue

What are dextrans? Colour Molar mass

~ 2 × 106 g/mol

Potassium chromate

formula:

Colour Molar mass

g/mol

There is a chromatographic column filled with Sephadex gel on the working place. Sephadex is polysaccharide of dextran type.

Procedure:

(1) Uncap the chromatographic column, open the tap and allow carefully run out the excess of fluid until the surface of the gel bed is reached. Pipette on the gel slowly 0.5 ml of the coloured mixture (potassium chromate and dextran blue). Use the pipette with fixed volume (=500 µl) and proper tip (blue). Pipette carefully! Not to whirl the surface of the gel!

Colour of the mixture:

(2) By turning the tap let the sample pass into the column.

(3) Pipette (using glass pipette and rubber suction bulb) about 3 ml of elution solution (physiologic solution; 0.9% NaCl) and let it penetrate the gel in the same way.

(4) Elute the column with sufficient amount of elution solution until both fractions leave the outflow.

(5) Collect both fractions in calibration test tubes.

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What is happening in the columne? Describe the changes:

Fraction order Colour Substance Fraction volume

1

2

Explain the order of separated dyes:

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β-Carotene – extraction and estimation

Extraction procedure:

(1) For disintegrating the tissue put 1g of grated fresh carrot into a mortar and grind it carefully using a pestle. Add 20 ml of 96% ethanol and mix the content. The suspension is then quantitatively transferred on a cellulose filter, and washed with acetone collected in a low-lying vessel until the sediment turns completely colorless.

(2) The acetone filtrate is then poured into a separatory funnel. Make sure that the lower stopcock of the funnel is closed. Add 40 ml of petroleum ether (benzine) and in addition water to fill the funnel to the ¾ of the total volume. Gently swirl the funnel for about a minute to allow carotene entering the upper petroleum ether (p.e.) layer. Place the funnel in a ring stand, remove the tap, and wait until the two layers are fully separated. Open both top and bottom taps and release the lower water phase. This liquid is to be discarded.

(3) Add 20ml of 20% ethanol to the funnel and adjust the volume again to ¾ of the total with water. Gently swirl the funnel again and repeat the washing 2-3 more times, until the ethanol-water phase remains colorless. Transfer the p.e. extract to a bottle equipped with a stopper, add a spoonful of waterless sodium sulfate and shake the content for 20 s.

(4) Clarify the extract by filtration into a 50 ml volumetric flask. Combine with the filter and salt p.e. wash before filling the flask with p.e. up to the mark. Mix, and put an aliquott to the photometric cuvette. Read the optical density (absorbance) at 450 477 nm. The beta-carotene content (mg/kg of carrot) is determined from the calibration graph. The beta-carotene purity is assessed by the difference between absorbance values at 450 and 477 nm. For a pure beta-carotene, the proportions of the two values ranges between 1.16-1.18.

Note: The separatory funnel runs on the concept of "like dissolves like". This means that if a compound is polar, then it can only be dissolved in a polar solvent. Following this logic, a

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non-polar compound can only be dissolved in a non-polar solvent. In a separatory funnel, there are two phases: the aqueous layer is polar, and the organic layer is non-polar. Because these two layers share a surface, any polar substances that were initially in the solution will be pulled into the polar/aqueous layer, and any non-polar substances will be pulled into the non-polar/organic layer. The mixtures to be separated are added through the top with the stopcock at the bottom closed. The funnel is then closed and shaken gently by inverting the funnel multiple times, temporarily releasing the upper tap to reduce the vapor pressure. Following the shaking, the separating funnel is set aside to allow for the complete separation of the phases. The denser of the two layers is then removed via the stopcock. Biological value of carotenoids and vitamin A:

Carotenoids are yellow, orange, or red tetraterpenes (40C). Carotenoids are found in plants and are known as provitamin A, as they can be cleaved to yield retinaldehyde and hence retinol and retinoic acid, collectively called vitamin A. The alpha-, beta- and gamma-carotenes are the most important provitamin A carotenoids. These carotenoids are cleaved in the intestinal mucosa by carotene dioxygenase, yielding retinaldehyde, which is reduced to retinol. The intestinal activity of carotene dioxygenase is low, so that a relatively large proportion of ingested beta-carotene may appear in the circulation unchanged and beta-carotene is only one-sixth as effective a source of vitamin A as retinol. However, beta-carotene, unlike vitamin A, is an antioxidant due to its conjugated double bonds.

Vitamin A plays an important role in vision. In retina, retinaldehyde functions as prosthetic group of the light-sensitive opsin proteins, forming rhodopsin in rods. In vitamin A deficiency, both the time taken to adapt to darkness and the ability to see in poor light are impaired. A more prolonged deficiency leads to xerophthalmia: keratinization of the cornea and skin and blindness. Vitamin A is toxic in excess, affecting the central nervous system, the liver, the bones and the skin.

Retinoic acid regulates growth, development, and tissue differentiation. Like the steroid hormones regulate the transcription of specific genes.

Molecular biology 1 DNA isolation

Isolation of genomic DNA from buccal swabs - a brief protocol

MACHEREY-NAGEL isolation kit

Protocol:

1. Gently rub and rotate swab along the inside of the cheek (both left and right side), ensuring that the entire swab tip has made contact with the cheek, approx. 2-3 min. During removing the swab from mouth, be careful not to touch swab tip against teeth, lips, or other surface.

2. Put the swab tip into 1.5mL eppendorf tube and break the wooden stem so that the part with cotton stays in the eppendorf tube.

3. Add 100 µL PBS, 15 µL proteinase K, 100 µL Buffer B3 . Vortex vigorously for 60s. 4. Put the tube into thermoblock and incubate at 56°C for 10 min. Vortex for 30s. 5. Put the tube into thermoblock and incubate at 70°C for 5 min. Vortex for 30s. 6. Add 100 µL 96% ethanol and vortex for 10s. Spin down shortly. 7. Load the lysate (without cotton part of the forensic swab) onto the NucleoSpin® Column.

Centrifuge 1 min at 12,000 RPM. Discard Colection Tube with flow-through. 8. Place the NucleoSpin® Column into a new Collection Tube and add 400 µL Buffer BW .

Centrifuge 1 min at 12,000 RPM. Discard Colection Tube with flow-through. 9. Place the NucleoSpin® Column into a new Collection Tube and add 400 µL Buffer B5

Centrifuge 1 min at 12,000 RPM. Discard the flow-through and reuse Collection Tube. 10. Place the NucleoSpin® Column back into the Collection Tube and centrifuge 3 min at 12,000 RPM. 11. Place the NucleoSpin® Column in a new 1.5mL eppendorf tube and add carefully 50 µL preheated

Buffer BE (70°C). Dispense the buffer directly onto the silica membrane! Incubate at room temperature for 1 min.

12. Centrifuge 1 min at 12,000 RPM.

Assessment of DNA concentration and purity

There are several methods to determine the concentration of nucleic acids as well as their purity -

spectrophotometry (absorbance measurement), agarose gel electrophoresis, fluorometry using fluorescent

DNA-binding dyes

In our practical classes, we will use spectrophotometric estimation. Nucleic acids absorb ultraviolet light

with an absorption maximum at 260 nm, proteins at 280 nm. Low molecular weight substances (eg

phenol, chloroform, EDTA, polysaccharides ...) have its absorption maximum at 230 nm. Absorbance at

320 nm indicates the presence of undissolved solid particles or a contaminated cuvette.

The nucleic acid concentration is calculated from the absorbance measured at 260 nm.

A260 = 1 corresponds to:

• double stranded DNA (dsDNA) at concentration 50 µg/ml • single stranded DNA (ssDNA) at concentration 37 µg/ml • RNA at concentration 40 µg/ml

Molecular biology 1 DNA isolation

The ratios A260/A280 a A260/A230 is used to assess the purity of nucleic acids. The ratio A260/A280 should be

for pure DNA around 1.8, for pure RNA around 2. Lower A260/A280 values may indicate protein

contamination. The ratio A 260/A230 should be for pure DNA higher than 2.0. Lower A260/A230 values

indicate contamination with salts or solvents, such as phenol. Residual chemical contamination from

nucleic acids extraction procedures may result an overestimation of the nucleic acid concentration and/or

negatively influence downstream analysis. If the required purity is not met, reprecipitation of the sample is

required, resulting in a significant reduction in the impurity content.

Protocol:

DeNovix DS-11 microvolume Spectrometer - apply 1 µl of sample.

1. Select the program: "dsDNA" on the spectrophotometer.

2. Set the Spectrophotometer against the blank solution ("blank") - in our case BE Buffer:

Ensure both top and bottom sample surfaces are clean. Pipette 1 µl of BE Buffer onto the lower sample surface, lower the top arm and press the BLANK button. Then open the lid, remove the solution from both sample surfaces using a clean, dry lab wipe.

3. Measure the absorption spectrum of the sample within the range 220 nm - 350 nm:

Pipette 1 µl of sample onto the lower sample surface, lower the top arm and press the MEASURE button. Then open the lid, remove the solution from both sample surfaces using a clean, dry lab wipe.

4. The device displays the measured spectrum, DNA concentration, and the A260/A280 and A260/A230 ratios that give us information about the purity of the solution.

5. Print an overview of the measured values.

A) Pipetting sample solution onto pedestal. B) Measurement.

Molecular biology 2 Polymerase chain reaction (PCR)

Polymerase chain reaction - a brief protocol

In this laboratory, we will amplify a region of the Factor V gene (the region which contains SNP of our interest) using Polymerase Chain Reaction (PCR).

Wear gloves (powder-free)! Fill a polystyrene box with ice from the icemaker. Keep the reagents on ice always when possible.

Master Mix Preparation

The total volume for a single PCR reaction is very small (20 µL). It would be uncomfortable to pipette separately all the components needed for each PCR reaction. The strategy is to prepare so called Master Mix which contains all the common components for a set of reactions. It improves consistency among the reactions and reduces pipetting error.

Take a new 1.5mL eppendorf tube and prepare the Master Mix for 10 PCR reactions, each of the total volume 20 µL (19 µL Master Mix + 1 µL DNA sample).

µL

water 155 + 4 PCR buffer (10x) 20 Mg2+ 5 dNTP 4 forward primer (F) 2 reverse primer (R) 2 Taq polymerase 2 enzyme – freezer !

DNA sample 10 DNA sample will be added later into individual PCR test tubes ----------------------------------------------- 200 + 4

Vortex the eppendorf tube with Master Mix for 10s. Spin down shortly. Keep on ice.

Setting up PCR reactions

Take out your DNA sample from the freezer and let it melt at the room temperature.

Prepare 10 PCR test tubes. Dispense 19 µL of the Master Mix into each tube. Add 1 µL of the DNA sample. sample sample sample sample sample sample sample sample posit. neg. 1 1 2 2 3 3 4 4 control control Put the PCR tubes into the block of thermocycler and start up the predefined program. PCR Parameters: 1) Initial denaturation: 95°C - 5 min

2) 35 cycles of: 95°C - 15 s 60°C - 15 s 72°C - 30 s

3) Linked to: 72°C - 7 min

4) Linked to: 4°C - indefinitely

Molecular biology 3 Restriction enzyme analysis + gel electrophoresis + interpretation of the results

In this laboratory exercise, you will use the restriction endonuclease MnlI for analysis of the DNA fragment amplified by PCR method last time.

Wear gloves (powder-free)!

Restriction enzyme analysis

You will receive 3 PCR test tubes from the set of ten PCR reactions processed last time. There is 20 µL of PCR reaction mixture (hopefully with the PCR product) in each tube.

• your DNA sample test tube marked with your initials • positive control (factor V Leiden, heterozygote) test tube marked "+K"

• negative control test tube marked "–K"

To perform restriction enzyme analysis, take two new PCR test tubes and tranfer exactly 10 µL of the PCR product from the test tubes with your DNA sample and positive control into the new test tubes. Mark the new test tubes with symbol "R", i.e. "your initials R", "+KR".

Restriction analysis

10 µL 10 µL PCR products sample posit. neg.

control control The total volume of restriction analysis reaction will be 20 µL. Now there is 10 µL of the PCR product.

1. Add the following components in the order indicated:

PCR product (DNA) 10 µL water 7 µL 10x FastDigest Green Buffer 2 µL FastDigest enzyme MnlI 1 µL

--------------- 20 µL 2. Mix gently and spin down. 3. Incubate at 37° C in a heat block of the thermocycler for 10 min.

Molecular biology 3 Restriction enzyme analysis + gel electrophoresis + interpretation of the results

Gel electrophoresis Add 2 µL of loading dye into the three test tubes with PCR products. Load 10 µL of the reaction mixtures on a gel as demonstrated in the figure. There are 13 well in the gel we use. Each group of students will use one half of the gel (6 wells as indicated).

Interpretation of the results

length of PCR product: 288 bp restriction analysis: homozygote wild type: 158 bp, 93 bp, 37 bp

factor V Leiden heterozygote: 158 bp, 130 bp, 93 bp, 37 bp

factor V Leiden homozygote: 158 bp, 130 bp