University of NizwaSultanate of OmanCollege of Arts and Sciences

DEPARTMENT 0F BIOLOGICAL SCIENCE AND CHEMISTRY

Laboratory Manualin

Introduction to Biotechnology (BIOL 345)

Compiled by:

Dr. Syed Abdullah GilaniDr. Mostafa Abdulaziz Mansi

&Zahra Khalfan Al-Abri

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Preface

This laboratory manual is the compilation of the experiments which are necessarily needed for the students who will study Introduction to Biotechnology course. These experiments covers ranges of lab work from the very basic i.e., lab safety measures, preparing stock and working solutions, DNA extraction to specialized lab works Keeping in view all these points, these experiments are included into this manual.

The experiments included are those that illustrate the concepts that are taught in the lecture. The procedure and directions were made as simple for the students to understand and follow. The experiments are provided with questions as well as the concepts behind each step why we do certain steps during experiment using other materials like textbook.

This manual is still in its initial and experimental stage of use therefore, we would deeply appreciate and welcome any suggestions and comments for its improvement.

Dr. Syed Abdullah GilaniDr. Mostafa Abdulaziz MansiMs. Zahra Khalfan Al-Abri

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LAB NO. 1

LAB SAFETY RULES AND REGULATIONS

1. Never work alone in the laboratory.2. Always wear lab coats, gloves and proper eye protection when in the lab.3. Never perform any experiment not specifically assigned by your teacher4. Never eat, drink, or apply cosmetics in the laboratory.5. NEVER taste chemicals.6. It is best not to wear contact lenses in the lab. Chemical vapors can get between the lenses and the eyes and cause permanent eye damage.7. Know the location of all safety and emergency equipment used in the laboratory.8. Before beginning work: tie back long hair, roll up loose sleeves and put on any personal protective equipment required.9. Report any accidents, incidents, or hazards to the teacher immediately.10. Keep your work area neat and uncluttered.11. Clean your work area at the conclusion of a lab activity, disinfect your station with bleach solution.12. Wash your hands with antibacterial soap and water.13. Always respect lab work. Due to the amount of students that will utilize the biotech lab, there will be other experiments at or around the workstations. Please leave them alone.

Major hazards encountered in biotechnology laboratories• Microorganisms: most experiments are carried out using E. coli which are unable to

survive outside the test tube. Nevertheless precautions should be taken when handling the bacteria. E. coli is chosen because it is easily handled and it has a number of cloning vectors specific for it.

• Radiochemicals: 35P and 32S are dangerous radionuclides. Care should be taken when dealing with such chemicals. They should be handled with care and disposed at the specific place.

• Organic solvents: most dangerous solvent used is phenol. Always gloves should be worn when carrying out phenol extraction.

• Mutagens and carcinogens: most chemicals that bind to DNA in a test tube are mutagenic and carcinogenic. Most toxic mutagenic and carcinogenic is Ethidium bromide.

• Toxic chemicals: the most dangerous toxic chemical is Acryl amide because of the possibility of creating aerial dust contamination when handling the powder.

• Ultraviolet radiation: Most dangerous equipment is the Ultra violet transiluminator.• High voltage Electricity: usually the voltage used in electrophoresis can kill. Broken

tanks should not be used.General Safety Precautions in Handling Hazardous Chemicals in the LabThere are generally four routes to exposure to hazardous chemicals that you should keep in mind while handling them: Inhalation: avoid by the use of fume hoods and masks Skin & eye contact: avoid by the use of lab coats, gloves, and goggles Ingestion: avoid eating or drinking in the lab or leaving the lab without removing gloves

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and washing hands Injection: dispose of broken glass and needles properlyBecause chemicals pose so many different kinds of hazards, there are no simple rules of thumb for safe handling of them all except for some common sense measures: Treat all chemicals as if they were hazardous until you learn otherwise Label all containers with contents, including concentrations and date that they were transferred If a hazardous material is contained, label it with a warning Think through your experiment BEFORE doing it, making sure that you will not be combining incompatible chemicals Clean your bench top before and after use Wash hands often and ALWAYS before leaving the lab Take off lab coats and gloves before leaving the lab Always remove gloves before touching phones, doorknobs, light switches, etc. Ensure proper waste disposal and sopropan.Here are some specific tips for handling the different types of hazardous chemicals: Flammables: Do NOT heat these reagents unnecessarily, and never in the presence of a flame or source of a spark. In general, only open containers in fume hoods. When storing more than 10 gallons of flammable liquids, a special explosion proof storage cabinet is required. Corrosives: Wear personal protective equipment (PPE) such as lab coats, goggles and gloves, and always add strong acids or bases to water when making solutions. Neutralize slowly to avoid rapid generation of heat and gases. Strong acids and bases should never be stored together. Reactive chemicals: Wear PPE such as lab coats, goggles and gloves, and know the reactive properties of the chemical. Always store oxidizing chemicals away from flammable materials. Toxic chemicals: Wear PPE such as lab coats, goggles and gloves, and know the toxic properties of the chemical. When working with a dry powder, wear a mask to avoid breathing the dust. Be aware of the waste disposal procedures for unused reagents and materials that come in contact with the chemical.

Here are some of the most common hazardous chemicals that you will encounter in the biotechnology lab:Carcinogens – formaldehyde Mutagens – ethidium bromideNeurotoxins – acrylamide Teratogens – formamideNephrotoxins – acetonitrile Hepatotoxins – chloroformCorrosives – phenol, strong acids & bases

Limit access to the lab at the discretion of the lab director, and adequately train all lab personnel. Use personal protective equipment (PPE) at all times, and keep all PPE inside the lab. Wash hands after handling viable materials and animals, after removing gloves and before leaving the lab. Always remove gloves before touching phones, doorknobs, light switches, etc.

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Avoid touching your face with your hands or gloves. Keep personal items such as coats and book bags out of the lab or in a designated work area.No mouth pipetting; use mechanical pipetting devices. Minimize splashes and aerosol production. Disinfect work surfaces to decontaminate after a spill and after each work session. Disinfect or decontaminate glassware before washing. Decontaminate all regulated waste before disposal by an approved method, usually by autoclaving. Have an insect and rodent control program in effect. Use a laminar flow biological safety cabinet when available.Seventy percent of recorded laboratory-acquired infections are due to inhalation of infectious particles, so special precautions should be taken to avoid producing aerosols when working with pathogens. While performing activities that mechanically disturb a liquid or powder, the biotechnologist should make the following adjustments.

Activity Shaking or mixing liquids mix only in closed containers Pouring liquids pour liquids slowly Pipetting liquids use only cotton plugged pipettes Removing a cap from a tube point tubes away when opening Breaking cells by sonication in the open sonicate in closed containers Removing a stopper or cotton plug remove slowly Centrifuging samples use tubes with screw cap lids Probing a culture with a hot loop cool loop first

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LAB NO. 2

PREPARATIONS OF STOCK SOLUTIONS

Stock Solutions (Autoclave then all reagents)

Buffers for DNA extraction and DNA electrophoresis

Tris Boric EDTA (TBE): Recipe for 1 litre (10x stock):

• 108g Tris base

• 55g boric acid

• 7.3g EDTA pH=8.2-8.4

Tris acetate EDTA (TAE): Recipe for 1 litre (10x stock):

• 48.4g Tris base

• 11.4 glacial acetic acid

• 20ml of 0.5M EDTA (pH=7.6)

Tris EDTA (T.E): Recipe for 1 litre (10x stock):

• 10mM Tris HCl

• 1mm EDTA (pH=8).

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Extraction of DNA

Equipment and reagents

• Phenol

• TE buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0)

• 24:1 (v/v) chloroform-isoamyl alcohol

• 3 M potassium acetate, pH 5.5, prepared by adding glacial acetic acid to 3 M potassium acetate until this pH is obtained

• Microcentrifuge

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LAB NO. 3

DNA EXTRACTION FROM PLANTS LEAVES/STEM/ROOTS/MICROBES

Method

1 Add an equal volume of phenol to the DNA containing reaction mixture and vortex gently.

2 Separate the aqueous phase which contains the DNA from the organic phase by centrifugation in the microfuge, at 2 000 rpm for 5 min or at 8 000 rpm for 1 min.

3 Remove the aqueous phase with care into a fresh microfuge tube and add an equal amount of 24:1 (v/v) chloroform-isoamyl alcohol.

4) In order to precipitate the DNA, add a 0.1 volume of 3 M sodium acetate, pH 5.5, to the aqueous phase and then 2 volumes of absolute ethanol. Incubate at –20 °C overnight or for shorter periods at –80 °C (e.g. 20–30 min).

5) Recover the precipitated DNA by centrifugation in the microfuge at 10 000 rpm for 5–15 min. Remove the ethanol with care and dry the pellet in a desiccator or 50 °C oven for 5 min. An extra wash with 70% (v/v) ethanol may be included to remove excess salt from the pellet. The dried DNA may be resuspended in sterile TE, pH 8.0, or water, and stored at 4 °C for further manipulation or at –20 °C for long-term storage.

DNA Extraction from Bacteria

Materials:

E. coli suspension (Luria Broth, 37oC overnight) 1% Sodium Dodecyl Sulfate (SDS)Test tubes Water Bath (60-65oC)Stirring rod 95% Ethanol (chilled)Dropper

Safety: Be careful working with Bacterial culture! Wash hands!

Procedure:

1) Add 5 ml. E. coli suspensions to a test tube.

2) Add 1 ml. of SDS to the Tube with E. coli, gently rotate and swirl to mix in SDS approx. 5 min.

3) Stand tube in a 60-65o C water bath for 30 min.

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4) Allow to cool to room temp.

5) Place a stirring rod into the test tube and add 2 ml. of cold 95% ethanol slowly with the dropper down the stirring rod into the tube.

6) Hold tube at a 45 degree angle and slowly rotate your stirring rod clockwise (avoid touching sides of tube) approx. 5 min.

7) Remove rod and immerse rod with bacterial DNA in 95% ethanol for approx. 2 min.

Bromophenol Blue (Loading Dye)

Makes 100 ml. Store at room temperature (indefinitely).

• 0.25 g bromophenol blue (M.w. 669.96)

• 0.25 g xylene cyanol (M.w. 538.60)

• 50.00 g sucrose (M.w. 342.30) (or 50 ml of glycerol)

• 1.00 ml 1M Tris (pH 8.0)

If using sucrose:

• Dissolve bromophenol blue, xylene cyanol, sucrose and Tris in 60 ml deionized or distilled water.

• Add deionized or distilled water to make 100 ml total solution.

If using glycerol:

• Dissolve xylene cyanol, bromophenol blue, and Tris in 49 ml of deionized or distilled water.

• Stir in 50 ml of glycerol to make 100 ml total solution.

CTAB and SDS: For plant cells with a rigid cell wall, the disruption of cells usually requires the tissue to be ground using a pestle and mortar in a pool of liquid nitrogen. The powdered plant tissue is then transferred to an extraction buffer that contains detergent to disrupt the membranes. SDS and CTAB are commonly used for this purpose. SDS has the advantage of denaturing proteins that can be precipitated in the presence of high salt (e.g potassium acetate) and subsequently can be pelleted and removed.

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Ethylenediaminetetraacetic acid (EDTA) a chelating agent and 2-mercaptoethanol or DTT) a reducing agent: These help to inactivate nucleases that are released from the plant cell and can case serious degradation of the genomic DNA. Their effects can be minimized by keeppeng the reaction cold, when possible.

Isopropanol or Ethanol: Phenol extraction or alternative resin purification methods can be used to remove any traces of protein and the genomic DNA precipitated using either ethanol or Isopropanol.

Soak mortar and pestle in detergent overnight, wash next day, sterilize at 160°C for 16 hours in oven.

RNAse Treatment

1. Add Rnase 2 ul (10ng/ml) of RNAse, Incubate for 1 hour at 37 °C. (Optional: Add 1/10 volume of 3 M Sodium acetate)

2. Dilute with 2 X volumes (200 ul) dDW, add 300 ul chloroform:isoamylalcohol. Mix gently and thoroughly.

3. Centrifuge 14,800 rpm room temperature for 10 minutes, Collect aqueous phase to new tube.

4. Add 2.5 X volume of 100 % Ethanol5. Spin down at 14,000 rpm for 10 minutes and discard6. Wash with 70 % ethanol and dissolve in water

OR:

1. Add 1 µl Rnase in 100 μl DNA solution and incubate at 37 °C for 30 minutes. 2. Add 100 μl MilliQ.3. Add 50 μl of 10M Ammonium Acetate (should be 7.5 M stock with Final conc. 2.5 M).

(using M1V1 = M2V2 ---- V1 = 2.5 M x 200 μl /10M === 50 μl).4. OR Add Rnase 2 µl (10 ng/ml) of RNAse, Incubate for 1 hour at 37 °C. (Add 1/10

volume of 3 M Sodium acetate) 5. Add 2.5 X volume of 100 % Ethanol (625 μl)6. Spin down at 14,000 rpm for 10 minutes and discard7. Wash with 70 % ethanol, centrifuge at 10,000 rpm for 5 minutes, dry the pellet and

dissolve in water.

Details of protocol for RNAse Treatment

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1. Make DNA upto 100 μl. Add RNAse 1 μl into it.2. Incubate at 37 ºC for 30 minutes.3. Add dDW 125 μl and mix.4. Add Ammonium Acetate (from 10M) 75 μl and mix.5. Add cold ethanol 750 μl. Mix gently.6. Spin down at 14,000 rpm for 10 minutes. Discard ethanol.7. Add 0.5 – 1 ml Wash buffer OR 70 % ethanol.8. Keep atleast for 15 minutes at room temperature.9. Spin down at 14,000 rpm for 10 minutes. Discard the solution and Dry it.10. Add dDW.

Safety Measures and Waste Handling

1. Always wear gloves to avoid risk hazards of toxic chemicals.2. Don’t throw any chemical/reagent waste into the sink.3. Use fumehood while using HCl, mercapthoethanol, chloroform-isoamyl alcohol, phenol

etc.4. If beaker or flask was used for concentrated chemical reagents, e.g., Chloroform, phenol

etc., washing directly into sink is prohibited. Rinse three times and throw the rinsed water in waste tank first and then wash it in a sink.

5. Adjust pH of alkaline or acidic solutions first, discard in its waste bottle if contains any halogen, chloroform, phenol particles, then rinse the glassware three times and throw in a waste tank and then wash in a sink.

6. Ethidium bromide: It is highly carcinogenic. Wear surgical gloves on both hands. Then wear the plastic glove on one hand. The other hand, with surgical glove only, should deal with opening closing doors and handling general matters. But the hand with plastic glove should touch only the ethidium bromide-used box inside and taking and handling agrose gel-soaked-in-ethidium bromide.

7. Chloroform-Phenol containing Tubes/Tips: When the pellet in chloroform remains in eppendorf tubes, don’t discard the tubes directly into specialized waste bin for chloroform/phenol materials. First filter the material, separate the liquid, discard it into the waste bottle of chloroform/phenol. And then discard the hard material into waste bin. Don’t leave chloroform containing tips in the waste bin on your table top, it causes smell all around. Also don’t leave the tips used for mercaptoethanol at your bench top waste bin.

2-Mercaptoethanol

2-mercaptoethanol is a reducing agent which has been shown to enhance growth 1, plating efficiency 2 and/or antibody synthesis 3 of specific cell lines. The kinetics of this phenomenon is not fully understood. Most applications do not require supplementation with 2-mercaptoethanol. Store unused portions of 2-mercaptoethanol tightly capped in the original container.

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Cetyltrimethylammonium bromide (CTAB) buffer:

For plant cells

General: Cetyltrimethylammonium bromide (CTAB) is a cationic detergent that has the useful property of precipitating nucleic acids and acidic polysaccharides from solutions of low ionic strength. Under these conditions, proteins and neutral polysaccharides remain in solution. In solutions of high ionic strength, CTAB forms complexes with proteins and all but the most acidic polysaccharides, but will jot precipitate nucleic acids. CTAB is therefore, particularly useful for purification of DNAs from organisms that produce large quantities of polysaccharides e.g. plants, and certain Gram-negative bacteria (including some strains for E. coli). CTAB is used in two types of basic precipitation procedures.

a. For preparation of genomic DNAs: The detergent is added to bacterial or cell lysates that have been adjusted to high ionic strength (>0.7 M NaCl). After removing the CTAB/polysaccharides/protein complexes by sequential extraction with chloroform and phenol, the genomic DNA is recovered from the supernatant by precipitation with sopropanol or ethanol.

b. For preparation of phaemid, plasmid and bacteriophage DNAs: CTAB is added to lysates of low ionic strength. The precipitated DNAs are collected by centrifugation, dissolved in solutions of high ionic strength and purified by ethanol precipitation.

CTAB and other cationic detergents also have the remarkable property of enhancing the rate of renaturation of complementary DNA strands. At a concentration of I mM CTAB, renaturation rates can be as much as 10000 time faster than those in water. This rate is ~2000 times faster than that obtained in a 1 M solution of NaCl. The annealing reaction in the presence of CTAB is second order with respect to DNA concentration, and the rates approach those with which two complementary strands collid in a solution. CTAB also stabilizes the double-stranded DNA helix once formed. Annealing reactions remain rapid in the presence of as much as a 106-fold excess of noncomplementary DNAs. Although not widely publicized, it seems likely that CTAB or another cationic detergent is the active ingredient in the numerous “rapid hybridization” solutions that are commercially available.

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LAB NO. 4

GEL ELECTROPHORESIS

Bromophenol Blue (Loading Dye)

Makes 100 ml. Store at room temperature (indefinitely).

• 0.25 g bromophenol blue (M.w. 669.96)

• 0.25 g xylene cyanol (M.w. 538.60)

• 50.00 g sucrose (M.w. 342.30) (or 50 ml of glycerol)

• 1.00 ml 1M Tris (pH 8.0)

If using sucrose:

• Dissolve bromophenol blue, xylene cyanol, sucrose and Tris in 60 ml deionized or distilled water.

• Add deionized or distilled water to make 100 ml total solution.

If using glycerol:

• Dissolve xylene cyanol, bromophenol blue, and Tris in 49 ml of deionized or distilled water.

• Stir in 50 ml of glycerol to make 100 ml total solution.

Gel Electrophoresis

To prepare 1% Agarose Gel:

• Weigh out 1g of Agarose into a flask and add 100ml of 1 x TBE or TAE.

• Heat solution in a microwave or boiling water bath until Agarose is completely dissolved.

Allow to cool in a water bath set at 50 – 55 °C for 10 min.

• Prepare gel casting tray by sealing ends of gel chamber with tape or appropriate casting system.

• Place appropriate number of combs in gel tray.

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• Pour into gel tray. Allow to cool for 15-30 min at room temperature. Gels can also be placed in a cold space and used the following day.

• Remove comb (s), place in electrophoresis chamber and cover with buffer (TAE or TBE as used previously).

• Add loading buffer to samples. As a guideline, add 1.5 ul of 10x Loading Buffer to a 20-25 ul PCR/DNA solution.

• For more concentrated DNA solutions (e.g. plasmids), prepare tubes with 8 ul of 2x Loading Buffer and 2 ul of plasmid.

• Load DNA and standard (Ladder) onto gel.

Electrophorese at 100V for 1 h.

• Visualize DNA bands using UV light box or gel imaging system.

Detection of DNA/RNA using Ethidium Bromide

CAUTION: ETHIDIUM BROMIDE IS A POTENT MUTAGEN. HANDLE ONLY WITH GLOVES AND PROPER PRECAUTIONS.

Method I - Including Ethidium bromide in the gel and buffer

1. Dissolve Agarose in buffer using the standard protocol for gel preparation.

2. Allow gel to cool to 60-70°C.

3. Add Et Br to 0.5 µg/ml final concentration. (Stocks are generally 10 mg/ml, and require 5µl stock/100ml gel). Pour gel and allow to set as usual.

4. 5) Prepare enough buffer to fill the apparatus.

5. 6)Then run the gel.

6. 7) Upon completion of the run, place gel in plastic wrap on a UV light box. Bands will appear bright orange on a faint orange background.

7. 8) This method will detect approximately 5ng of DNA. Destaining in water or 1 mM MgSO4 may be required to achieve full sensitivity.

Method II - Post Run Staining

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1. Prepare enough 0.5µg/ml EtBr in water or buffer to completely submerge the gel. This solution is stable for 1-2 months at room temperature in the dark.

2. After the run submerge the gel in the staining solution for 15-30 minutes (depending upon gel thickness).

3. Place the gel on plastic wrap on a UV light box and observe under 300nm illumination. Bands will appear bright orange on a pale orange background.

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LAB NO. 5

TO DETERMINE DNA CONCENTRATION, YIELD AND PURITY USING UV-SPECTROPHOTOMETER

DNA yield can be assessed using various methods including absorbance (optical density), agarose gel electrophoresis, or use of fluorescent DNA-binding dyes. All three methods are convenient, but have varying requirements in terms of equipment needed, ease of use, and calculations to consider.

Absorbance Methods

The most common technique to determine DNA yield and purity is measurement of absorbance. Although it could be argued that fluorescence measurement is easier, absorbance measurement is simple, and requires commonly available laboratory equipment. All that is needed for the absorbance method is a spectrophotometer equipped with a UV lamp, UV-transparent cuvettes (depending on the instrument) and a solution of purified DNA. Absorbance readings are performed at 260nm (A260) where DNA absorbs light most strongly, and the number generated allows one to estimate the concentration of the solution. To ensure the numbers are useful, the A260 reading should be within the instrument's linear range (generally 0.1–1.0).DNA concentration is estimated by measuring the absorbance at 260nm, adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50µg/ml pure dsDNA.Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/mlTotal yield is obtained by multiplying the DNA concentration by the final total purified sample volume.DNA yield (µg) = DNA concentration × total sample volume (ml)However, DNA is not the only molecule that can absorb UV light at 260nm. Since RNA also has a great absorbance at 260nm, and the aromatic amino acids present in protein absorb at 280nm, both contaminants, if present in the DNA solution, will contribute to the total measurement at 260nm. Additionally, the presence of guanidine will lead to higher 260nm absorbance. This means that if the A260 number is used for calculation of yield, the DNA quantity may be overestimated.To evaluate DNA purity, measure absorbance from 230nm to 320nm to detect other possible contaminants. The most common purity calculation is the ratio of the absorbance at 260nm divided by the reading at 280nm. Good-quality DNA will have an A260/A280 ratio of 1.7–2.0. A reading of 1.6 does not render the DNA unsuitable for any application, but lower ratios indicate more contaminants are present. The ratio can be calculated after correcting for turbidity (absorbance at 320nm).DNA purity (A260/A280) = (A260 reading – A320 reading) ÷ (A280 reading – A320 reading)Strong absorbance around 230nm can indicate that organic compounds or chaotropic salts are present in the purified DNA. A ratio of 260nm to 230nm can help evaluate the level of salt carryover in the purified DNA. The lower the ratio, the greater the amount of thiocyanate salt is present, for example. As a guideline, the A260/A230is best if greater than 1.5. A reading at 320nm

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will indicate if there is turbidity in the solution, another indication of possible contamination. Therefore, taking a spectrum of readings from 230nm to 320nm is most informative.

How to Measure:

1. Add pure TE buffer or double distilled water (dDW) in a cuvet and measure the reading. Note: if the DNA is dissolved in TE buffer after extraction, then use TE buffer for spectrophotometry and if it was dissolved in dDW, then use dDW for spectrophotometer method.

2. Add 1 µl DNA in 99 µl TE buffer or dDW (use always 1:99 ratio, if the cuvet is large, then use 3 ml (30 µl DNA + 2970 dDW). Measure the reading on UV-Spectrophotometer using DNA concentration method (Bio-Method).

3. Note the DNA concentration and readings for 260 nm, 280 nm, and 230 nm.

4. Calculate the ratio 260/280 nm, if it is in the range of 1.8 – 2.0, the DNA would be of good quality, below this and above this value, there would be contamination in it.

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LAB NO. 6

POLYMERASE CHAIN REACTION PROTOCOL

Set up PCR tubes.

• Place thin-walled PCR tubes on ice.

• For a 50 μL reaction, add:

• 2 μL Template DNA (10 ng-500 ng)

• 5 μl10X Taq buffer with MgCl2

• 1 μldNTP mix (10 mM each nt)

• 2.5 μLForward Primer (10 μM stock)

• 2.5 μLReverse Primer (10 μM stock)

• 0.2 μLTaq DNA Polymerase (5 units/μL)

• 32.8 μLSterile deionized water (variable

Tip: If you are doing multiple PCR reactions, save time by creating a "master mix."

The following is a typical PCR program.

Step 1: Initial Denaturation for 2 minutes at 95oCStep 2: Denature for 1 minute at 95oCStep 3: Anneal primers for 30 seconds at 55oC (or 5oC below Tm)Step 4: Extend DNA for 2 minutes at 72oCStep 5: Repeat steps 2-4 for 25-30 cyclesStep 6: Final Extension for 10 minutes at 72oC

Run 2 μL on a gel to check size and concentration of PCR product

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Ethidium bromide stained gel showing PCR DNA fragments

LAB NO. 7

RESTRICTION ENDONUCLEASE DIGESTION OF PCR PRODUCTS

• 1µl 10x Buffer

• 6.5µl H2O

• 2µl DNA

• 0.5µl Enzyme

Incubate for 1 hour at 37°C in a water bath. Meanwhile, Run the fragmented DNA on Agarose gel.

Ethidium bromide stained gel showing DNA fragments

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LAB NO. 8

SDS POLYACRYLAMIDE GEL ELECTROPHORESIS

5X Sample Buffer (40ml)

ddH2O 16 ml

0.5 M Tris, pH 6.8 5 ml

50% Glycerol 8 ml

10% SDS 8 ml

2-βmercaptoethanol 2 ml (add immediately before use)

bromophenol blue

10% acetic acid

5X SDS Running Buffer (1 L)

Tris 15 g

Glycine 72 g

SDS 5 g

Coomassie Blue Stain (For proteins separation)

• 10% (v/v) acetic acid

• 0.006% (w/v) Coomassie Blue dye

• 90% ddH2O

Isopropanol Fixing Solution (For proteins separation)

• 10% (v/v) acetic acid

• 25% (v/v) isopropanol

• 65% ddH2O

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1x Running Gel Solution:

• For different applications increase your desired percentage acrylamide, make up thirty ml of running gel by selecting one of the following percentages and mixing the ingredients shown below.

• After adding TEMED and APS your gel will polymerize fairly quickly, so do not add these until you are sure you are ready to pour.

7% 10% 12% 15%

H2O 15.3ml

12.3ml

10.2ml

7.2ml

1.5MTris HCl, pH 8.8 7.5ml 7.5ml 7.5ml 7.5ml

20% SDS 0.15ml

0.15ml

0.15ml

0.15ml

Acrylamide/Bis-acrylamide

(30%/0.8%)

6.9ml 9.9ml 12.0ml

15.0ml

10% ammonium persulphate

0.15ml

0.15ml

0.15ml

0.15ml

TEMED 0.02ml

0.02ml

0.02ml

0.02ml

Stacking Gel solution (4% Acrylamide):

H2O 3.075ml

0.5M Tris-HCl, pH 6.8 1.25ml

20%SDS 0.025ml

Acrylamide/Bis-acrylamide 0.67ml

10% ammonium persulphate 0.025ml

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TEMED 0.005ml

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Pouring the Gels

Choose a percentage acrylamide based on the molecular weight range of proteins to be separated

% Gel M.W. Range

7 50-500 KDa

10 20-300 KDa

12 10-200 KDa

15 3-100 KDa

• Mix the ingredients needed for the chosen percentage and pour the solution quickly into the gel casting form - be sure to leave some room for the stacking gel (usually leave about 2 centimeters below the bottom of the comb for the stacking gel.

• You can do this by inserting the comb into the dry form, and marking a region below the comb for the height of the stacker you want).

• Look for bubbles and remove them, then layer the top of the gel with water saturated butanol or, very carefully, with water. This will remove bubbles at the top of the gel and will ensure this part does not dry out.

• Wait for about 30 minutes for the gel to polymerize completely. (If you always use fresh ammonium persulfate, you're gel may polymerize more quickly and reliably.)

• While waiting mix the reagents for the stacking gel, but LEAVE OUT the APS and TEMED until you are ready to pour this gel; stacking gels will polymerize more quickly than desired sometimes while one is trying to add combs to make wells.

• When the running gel is polymerized wash out the butanol completely or your stacker may separate from the gel and you will get ugly running artifacts.

• Mix in the polymerizing reagents and pour the stacking gel on top of the running gel.

• Insert your combs trying not to get bubbles stuck underneath and allow another 30 min - 1 hour for complete polymerization.

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Preparing your Sample:

• Mix your protein 4:1 with the sample buffer. Heat your sample by either:

a) Boiling for 5-10 minutes (Works for most proteins)

b) 65 degrees C for 10 minutes (If you have smearing using the above procedure)

c) 37 degrees for 30 minutes (Membrane proteins or others that do not enter the gel otherwise may benefit from this type of sample preparation)

Running the gel:

• Clamp in the gel and fill both buffer chambers with gel running buffer according to the instructions for the specific apparatus.

• Pipette your sample into the gel adjusting the volume according to the amount of protein in your sample.

• If you are going to stain using Coomassie, don't use much more than 5ug of your protein of interest to get a nicely defined band.

• Be sure to include a lane with molecular weight standards.

• Now attach your power leads and run the gel until the blue dye front reaches the bottom.

• I prefer to run at 250 V constant which in a four to twenty percent mini gel needs about 30 minutes total run time, but adjust to the thickness of your gel, the power supply used and the resolution desired.

• Remove the gel for the power supply and process further - Visualize your proteins using Coomassie Brilliant Blue, Silver stain, or any of the other protein stains.

• Use a carbohydrate stain for glycoproteins, or blot your gel for N-terminal sequencing or Western blotting.

Staining of gels with Coomassie blue:

• Place gel in a plastic container. Cover with isopropanol fixing solution and shake at room temperature. For 0.75 mm-thick gels, shake 10 to 15 min; for 1.5 mm thick gels, shake 30 to 60 min.

• Pour off fixing solution. Cover with Coomassie blue staining solution and shake at RT for 2 hr.

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• Pour off staining solution. Wash gel with 10% acetic acid to destain, shaking at RT ON.

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