basic molecular biology & biotechnology training manual

Basic Molecular Biology &

Biotechnology

Training Manual

I. The Molecular Design of Life 1. Prelude: Biochemistry and the Genomic Revolution 1.1. DNA Illustrates the Relation between Form and Function

GENETEC H LABORAT ORIES

Biotech Park in Biotechnology City, Lucknow

Genetech Laboratories

Sector - G, Jankipuram, Kursi Road, Lucknow – 226021 (U.P.) IndiaPh No: 0522-4006752 e-mail: [email protected]

Contents

S No Name of chapter Page No

1. List of Instruments 2

2. Abbreviations 3

3. Preparation of Buffers & Solutions 4

4. Bacterial Culture 7

5. Isolation of Plasmid DNA from Bacteria 9

6. Isolation of Genomic DNA from Bacteria 10

7. Isolation of Genomic DNA from Blood 12

8. Agarose Gel Electrophoresis 13

9. Quantification of DNA 15

10. Restriction digestion of DNA 16

11. Ligation of DNA fragments 17

12. Polymerase Chain Reaction 18

13. Elution of DNA from Agarose Gels 20

14. SDS PAGE 21

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1 - List of Instruments

1. Non Refrigerated Centrifuge

2. Refrigerated Centrifuge

3. Balance

4. BOD Incubator

5. Freezer

6. Gel Electrophoresis Unit

7. Micropipettes

8. Vortex

9. Dry Bath

10. Water Bath

11. Incubator Shaker

12. UV Transilluminator

13. Gel Documentation System

14. Water Purification System

15. Programmable Thermal Cycler

16. Laminar Air Flow

17. Gel Rocker

18. Magnetic Stirrer

19. Hot Plate

20. Spectrophotometer

21. pH Meter

22. Various Glasswares & Plasticwares

2 - Abbreviations & Symbols used

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DW : Distilled waterRT : Room temperatureg : grammM : mili molarM : Molar solutionN : Normal solutionml : mili literµl : micro literSDS : Sodium Dodecyl SulphateEDTA : Ethylene Diamine Tetra AcetateAPS : Ammonium PersulphateCBB : Commassie Brilliant BlueLB : Luria BertaniBPB : Bromo Phenol BlueTE : Tris EDTAnm : nano meterUV : ultra violetmol. wt : molecular weightEtBr : Ethidium BromideV : voltscm : centi meterbp : base pairskb : kilo baseOD : optical density

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3 - Preparation of Buffers &

Solutions

Solution I

a) Glucose 50mMb) Tris-Cl (pH 8.0) 25mMc) EDTA (pH 8.0) 10mMd) Autoclave and store at 4.0˚C

Solution II

a) NaOH 0.2Nb) SDS 1%

Solution III (for 50ml) or Potassium Acetate

(3M)

a) 5M Potassium Acetate 30mlb) Glacial Acetic Acid 5.75mlc) DW 14.25ml

Cell Lysis Buffer

a) 10mM Tris (pH=8.0)b) 1mM EDTAc) 0.1% SDS (w/v)

Gel loading dye

a) BPB 0.25%b) Xylene Cyanol 0.25%c) Sucrose 40.0%d) Store at 4˚C

TE (10X)

a) Tris-Cl (pH=8.0) 100mMb) EDTA (pH=8.0) 10mM

NaCl (5M)

To prepare 5M solution dissolve 292g of NaCl in 800ml of DW. Adjust the volume to 1 liter with DW. Dispense in aliquots, autoclave and store at RT.

Sodium Acetate (3M)

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Dissolve 408.3g of Sodium Acetate in 800ml DW. Adjust pH to 5.2 with Glacial Acetic Acid. Adjust the volume to 1 liter with DW. Dispense in aliquots and sterilize by autoclaving.

Ethidium Bromide

100mg EtBr is dissolved (with the help magnetic

stirer)in 10ml DW and stored in dark bottle at RT.

0.5M EDTA (pH 8.0)

Add 186.1g of Disodium Ethylene Diamine Tetra Acetate in 800ml DW. Stir vigorously on a magnetic stirrer. Adjust pH to 8.0 with NaOH. Make up to 1 liter by DW. Autoclave and store at RT.

TAE (50X)

Dissolve 242g of Tris in DW. Add 57.1ml of Glacial Acetic Acid and add 100ml of 0.5M EDTA (pH=8). Make up to 1 liter by DW. Autoclave and store at RT.

Resolving Gel: 15ml, 12.5%

a) DW 6.603mlb) Resolving gel buffer 1.875mlc) Acrylamide/bis-acrylamide (30:0.8) 8.33mld) 10% SDS 100µle) 10% APS 100µlf) TEMED 22µl

Resolving Gel Buffer (1.5M Tris, pH 8.8)

Dissolve 18.15g Tris base in 70ml DW. Adjust pH with 1N HCl. Make up to 100ml by DW and store at RT.

Stacking Gel: 5ml, 5%

a) DW 2.624mlb) Stacking gel buffer 1.25mlc) Acrylamide/bis-acrylamide(30:0.8) 1.0mld) 10% SDS 50µle) 10% APS 50µlf) TEMED 11µl

Stacking Gel Buffer (1.0M Tris, pH 6.8)

Dissolve 12.1g Tris base in 70ml DW. Adjust pH with 1N HCl. Make up to 100ml by DW and store at RT.

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Tris Glycine Electrode Buffer (pH 8.3)

a) Tris 3.0gb) Glycine 14.4gc) Dissolve in 800ml DW, adjust pH to 8.3, make up

to 1000ml and store at RT

Acrylamide/bis-Acrylamide Solution

a) Acrylamide 30gb) Bis-acrylamide 0.8gc) Dissolve in DW, make up to 100ml and store at

4˚C

Ammonium Persulphate Solution (10%)

Dissolve 100mg of APS in 1ml DW and store at 4˚C. Prepare fresh solution every time.

SDS Solution (10%)

Dissolve 10g SDS in 100ml DW and store at RT.

Gel Staining Solution

a) Commasie Brilliant Blue 0.25%b) Methanol 45%c) Acetic Acid 10%d) DW 45%

First dissolve CBB in methanol and water then add Acetic Acid.

Destaining Solution

a) Methanol 45%b) Acetic Acid 10%c) DW 45%

Sample Preparation Buffer

a) 0.6M Tris-Cl (pH=6.8) 5mlb) SDS 0.5gc) Sucrose 5.0gd) β-Mercaptoethanol 0.25mle) 1% Bromophenol Blue 2.5mlf) Add DW to make up to 50ml and store at RT

6X Gel Loading Dye

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a) Bromophenol Blue 0.25% (w/v)

b) Xylene Cyanol 0.25% (w/v)

c) Sucrose 40.00% (w/v)

Store at 4oC

4 - Bacterial Culture

A culture of bacteria (E.coli) is normally received as a broth culture, on a Petri dish or as a freeze-dried culture. First of all a careful record of the strain no and genotype of the strain is maintained. By this, one can easily identify an appropriate medium and any additions such as antibiotics, which are necessary to ensure stability and maintenance of plasmids. It is necessary to dry the agar plates because E.coli is motile and it swims across the plate in the thin film of water, moreover contaminants also spread easily across the plate and desired single colonies cannot be isolated. To achieve this dry the plates overnight at 37˚C in an oven.

Protocol for preparation of Agar Platesa) Weigh 4% LB agar medium in a conical flask. Pour the desired

quantity of DW and mix it by swirling the flask.

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b) Boil in a microwave oven. Swirl the liquid to check that the agar is fully melted. Take care that agar does not boil over.

c) Autoclave and allow the medium to cool up to 45-50˚C. Add appropriate conc of antibiotic, stir to mix well and allow time for any air bubble to disappear.

d) Arrange the sterile Petri plates on a level surface and label the base of each plate to indicate the medium prepared.

e) Flame the neck of the flask containing medium and pour the required amount (approximately 10ml for short term bacterial culture) of medium into the plates.

f) Allow the plates to set. Dry the surface of the plates by overnight incubation at 37˚C. Check for contamination next morning and discard the plates with contamination.

g) Wrap the plates with parafilm and store the plates at 4˚C.

Single colony isolationThe principle of this technique is to streak a suspension of

bacteria until single cells are separated on the plate. Each individual cell then grows in isolation to produce a clone of identical cells known as a “Colony.” The majority of these cells are genetically identical. However, during growth, mutation of even a single colony can give rise to low levels of mutant cells. Repeated single colony isolations result in a pure culture.

Protocol (streak plate method)a) Flame a nichrome loop (3mm across and has 6cm stem).

Allow the loop to cool or cool by immersion in a sterile area of the medium.

b) Flame the neck of an overnight LB Broth culture and remove a loop of cells or pick a colony from overnight LB Agar culture.

c) Streak the cells at one side of a well-dried agar plate (in a manner to make the first arm of a pentagon). Streak several times close together.

d) Flame the loop and cool as before.e) Streak again, starting from one end to make second arm of

pentagon.f) Repeat the steps d) and e) to complete the pentagon.g) Label each plate to indicate the strain no, genotype of strain,

date of culture and name of person.h) Wrap the plates with parafilm.i) Incubate the plate at 37˚C with the medium facing

downwards to reduce the chance of droplets of condensation falling on the medium surface.

Examine the plates for colony morphology and the presence of possible contaminants. If all of the colonies are of uniform size and appearance it is assumed to be a pure culture. Subculture of almost any colony should give the required strain and the plate is worth keeping as a future source of a purified culture.

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Broth culture Protocola) Prepare 1.5% of LB broth medium in a conical flask.b) Autoclave and allow the medium to cool up to 45-50˚C. Add

appropriate conc of antibiotic, stir to mix well, allow time for any air bubble to disappear.

c) Arrange the sterile test tubes and label each test tube to indicate the strain no, genotype of strain, date of culture and name of person.

d) Flame the neck of the flask containing medium and pour the required amount (approximately 4-5ml ) of medium into the test tubes and fix cotton plug

e) Pick a single colony by a sterile toothpick from agar plates and culture the test tube.

f) Repeat step e) for as many test tubes as required.g) Place the tubes in an incubator shaker at 37˚C, 250rpm

overnight.

5 - Isolation of Genomic DNA from

Bacteria

IntroductionIsolation of intact high molecular weight DNA with sufficient

purity is a basic requirement for any genomic study. In fact, the application of molecular biology techniques to analyze genome depends on the ability to isolate pure, high molecular weight DNA. A protocol that works for a group may fail with others. Consequently, a number of DNA isolation methods have been developed for different target groups. Hence, protocol selected should be adequate, quick, simple, and cost effective and it should yield DNA

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reasonably pure and intact. The two factors that affect the integrity of DNA are mechanical shearing and nuclease activity. The former can be taken care by avoiding harsh treatment of lysates and taking measures to inactivate the endogenous nuclease activity can control the latter.

Bacteria are the simplest material to process and can easily be harvested by centrifugation. During the isolation of DNA from Bacteria the lysis buffer containing NaOH and SDS, Proteinase K is used for the removal of the RNase activity or extra RNA present in the extract. Phenol is very good solvent of proteins but can also dissolve small quantities of DNA, for this reason, it is preferable to use phenol chloroform on DNA sample which are not heavily contaminated because DNA does not dissolve in this organic mixture. DNA is purified and concentrated with ethanol and isopropanol. Sodium Acetate with SDS forms the network of the contaminating agents such as membranes, high mol wt RNA, proteins.

Reagentsa) 10% SDS (w/v)b) 20mg/ml Proteinase Kc) Phenol:chloroformd) Isopropanole) 3M Sodium Acetatef) TE

Protocola) Take 1.5ml of bacterial culture in a microfuge tube and spin

at 10000rpm for 3 min.b) Decant the supernatant and resuspend the pellet in 467µl TE

buffer by repeated pipetting.c) Add 30µl of 10% SDS and 3µl of 20mg/ml Proteinase K, mix

well and incubate for 2 hrs at 37˚C.d) Add an equal volume of Phenol:chloroform and mix well by

inverting, until the phases are completed mixed.e) Spin at 10000rpm for 5 min and transfer the upper aqueous

phase to a new tube.f) Repeat the step d) and e) for once.g) Add 1/10 volume of 3M sodium Acetate and 0.5 volume of

Isopropanol. Mix gently until the DNA precipitates.h) Centrifuge at 10000rpm for 5 min. Discard the supernatant

carefully and air-dry the pellete.i) Resuspend the pellet in 100µl TE buffer.j) Store at -20˚C.

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6 - Isolation of Plasmid DNA from

Bacteria

IntroductionPlasmid isolation is achieved by the disruption of cell wall by

alkali lysis. Bacteria are suspended in an isotonic solution of sucrose and after addition of EDTA, the cells are exposed to detergent and lysed by treatment with alkali. The treatments, which disrupt base pairing, cause the linear chromosomal DNA of the host to denature. However, the strands of closed circular plasmid DNA are unable to

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separate from one another because they are topologically interwined. When conditions (pH) are returned to normal, the strands of the plasmid DNA rapidly fall into perfect register and completely native super helical molecules are reformed. But, chromosomal DNA can not re-nature and forms a network consisting of chromosomal DNA/high mol. wt. RNA/SDS/Proteins/membrane complexes, which can be removed by centrifugation. Plasmid DNA is purified and concentrated with ethanol/isopropyl alchohol.

ReagentsProtocol

a) Prepare a culture of bacteria containing the plasmid with sufficient aeration and the appropriate antibiotic.

b) Transfer 1.5ml of the culture to a microfuge tube and centrifuge at 12000rpm for 1 min at 4˚C to form a pellet.

c) Decant the supernatant, and resuspend the cell pellet in ice-cold 0.2ml solution I. Ensure that a homogeneous suspension is formed.

d) Add 0.3ml freshly prepared solution II and invert the capped tube several times to mix the contents. The solution should be translucent and viscous. Store the tube on ice for 5 min.

e) Add 0.25ml of ice-cold solution III. Mix the contents by inverting the tube several times. Store the tube on ice for 5 min.

f) Centrifuge the bacterial lysate at 12000rpm for 5 min at 4˚C. Transfer the supernatant to a fresh tube.

g) Add and equal volume of phenol:chloroform. Mix the organic and aqueous phases, and then centrifuge the emulsion at 12000rpm for 2 min at 4˚C. Transfer the upper aqueous phase to a new tube.

h) Precipitate the plasmid DNA from the supernatant by adding 2 volumes of ethanol at RT. Mix and leave the tube at RT for 3 min.

i) Centrifuge at 12000rpm for 5 min at 4˚C.j) Discard the supernatant gently, and invert the tube on tissue

paper.k) Add 1ml of 70% ethanol to the pellet and invert the tube

several times. l) Centrifuge at 12000rpm for 3 min at 4˚C.m)Discard the supernatant and air dry the pellet.n) Dissolve the pellet in 100µl of TE (pH 8.0) containing 20µg/ml

DNase-free RNase. Mix the solution gently.o) Store at -20˚C.

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7 - Isolation of Genomic DNA from Blood

IntroductionProduction of great bio banks and DNA banks in national &

international levels is one of development pivots, which is very important in medical, agricultural, economical & forensic genetic fields. In this regard, one of the primary and important steps for all is DNA extraction with high quality & quantity in minimum time from cells. By using the following method, genomic DNA with high quality & quantity can be acquired in the shortest time. DNA prepared from blood as described in this protocol is suitable for use as a template in PCRs.

Reagentsa) Ethanolb) Isopropanol

c) Potassium Acetate (5M)

d)Cell lysis buffer

e)TE (pH=7.6)

f) Tris-Cl 20mM (pH=7.6)

g)DNase free RNase (4mg/ml)

h)Proteinase K (20mg/ml)

i) Blood

Protocola) Transfer 300µl of whole blood into two MCT.b) Add 900µl of 20mM Tris-Cl (pH=7.6) to each tube and invert

the capped tubes to mix the contents. Incubate the tubes at RT for 10min, occasionally inverting the tubes.

c) Centrifuge the tubes at 14500 rpm for 1min at RT.d) Discard all but 20µl of each supernatant.e) Resuspend the pellets of white cells in small amount of

supernatant left in each tube and repeat once from step b.f) Combine the resuspended cell pellet in a single tube and add

600µl of ice-cold cell lysis buffer. Homogenize the suspension quickly. (The SDS will precipitate from the ice-cold cell lysis buffer producing a cloudy suspension)

g) Add 3µl of Proteinase K solution to the lysate and incubate the digest for at least 3 hrs at 55oC.

h) Allow it to cool to RT and add 3µl of DNase free RNase. Incubate the digest for 30min at 37oC.

i) Allow the sample to cool to RT. Add 200µl Potassium Acetate solution and mix the contents of the tube by vortexing vigorously for 20sec.

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j) Centrifuge to pellet the protein/SDS complex at 14500rpm for 3min at 4oC. (If the pellet is not visible, incubate the lysate for 5min on ice and repeat the centrifugation step).

k) Transfer the supernatant to a fresh MCT and add 600µl Isopropanol. Mix the solution gently.

l) Recover the precipitate of DNA by centrifuging the tube at 14500rpm for 1.5min at RT.

m)Discard supernatant and add 600µl of 70% Ethanol to the DNA pellet. Invert the tube several times and centrifuge the tube at 14500rpm for 1min at RT.

n) Carefully discard supernatant and allow the DNA pellet to dry in air for 20min.

o) Dissolve the pellet of DNA in 100µl of TE (pH=7.6). To facilitate solubilization of DNA pellet incubate at 55oC for 30min.

8 - Agarose Gel Electrophoresis

IntroductionThis technique is repeatedly used in molecular biology to

separate DNA fragments and to assess quality and quantity of DNA samples.

PrincipleThe gel is made from Agarose, which is a copolymer of D-

galactose and 3,6-anhydro-L-galactose. It forms a gel by hydrogen bonding and the pore size of gel depends on the concentration of agarose in the gel. Low agarose concentration improves resolution of larger fragments but reduces resolution of smaller fragments and vice versa. Hence, concentration of agarose in the gel is decided according to the size range of DNA species to be separated.

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The gel is immersed in buffer and the DNA samples are loaded onto a well at one end of the gel and made to move through the gel by the application of electric current. Since, DNA is negatively charged hence will move towards the anode. However, the polysaccharide mix of the gel retards movement of the DNA by a process of sieving, so that small fragments move through faster and these fragments separate according to size.

The DNA samples are visualized by adding EtBr, a fluorescent molecule which intercalates within the DNA bases, extending the length of linear and nicked DNA molecules and making them more rigid. When EtBr is added, UV radiation at 254nm it is absorbed by the DNA and transmitted to the bound dye. The energy is re-emitted at 590nm in the red-orange region of the spectrum. EtBr is a powerful mutagen and hence the gel should be handled carefully with the gloves. The DNA bands can be visualized under UV and the data can be recorded by gel documentation system.

Characteristic features of gel electrophoresis are:a)The molecular weight of the DNA: the migration

rate is inversely proportional to the mol. wt.b)Agarose concentration: the migration rate is

inversely proportional to the agarose concentration.c) Conformation of the DNA: linear form travels

slowest and the supercoiled form travels fastest.d)Applied voltage: typical value is 5 V per cm2. The

heat generated during electrophoresis is dissipated by the buffer.

e)DNA being polynaionic at neutral pH, it migrates towards anode.

f) The loading dye for DNA contains glycerol (which gives density to help the samples sink to the bottom of the well) and marker dyes like Xylene Cyanol and bromophenol blue. BPB moves on par with 300-400bp and xylene cyanol with 2-3kb DNA

g)The DNA is visualized by adding EtBr, a fluorescent molecule which intercalates with the DNA bases. To 0.8% agarose gel add EtBr to a final concentration of 0.5µg/ml.

h) UV radiation at 254nm is absorbed by the DNA and transmitted to the bound dye. The energy is re-emitted at 590nm in a red-orange region of the spectrum.

i) EtBr is a powerful mutagen. The dye is usually incorporated into the gel or conversely the gel is stained after running by soaking in a solution of EtBr.

j) The usual sensitivity of detection is 0.01µg of DNA.

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k) The gel will be run along with a molecular weight marker, a wide range of which is commercially available.

Protocola) Take 1% (w/v) agarose in Tris-acetate EDTA buffer (1X

TAE) and heat to dissolve the agarose.b) Cool the solution to 45-50˚C and add EtBr (0.5 µg/ml).

Mix well.c) Before pouring the solution into gel casting tray wash

the tray and comb with DW, and place the tray on a leveled surface.

d) Choose appropriate comb and fix into position.e) Pour the gel into the apparatus and allow it to cool and

set.f) After the gel has set firmly, pour little amount of

buffer and remove the comb gently. Take care not to drag the comb and break the gel.

g) Immerse the gel slowly into the gel tank. Add sufficient amount of 1X TAE buffer. Connect the electrode and check the current.

h) Load the samples into wells carefully.i) Always load an aliquot of standard molecular weight

marker along with the samples. It will help in assessing the size of the DNA fragment by comparing with the electrophoretic mobility.

Anticipated ResultsDepending upon its conformation and size, the DNA

fragments will move as discrete bands. The EtBr intercalated with the DNA fragments will make it emit pink fluorescence under UV light.

9 - Quantification of DNA

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IntroductionReliable measurement of DNA concentration is important for

many applications in Molecular Biology including complete digestion of DNA by Restriction Enzyme and amplification of target DNA by PCR. DNA quantification is generally carried out by spectrophotometric measurements or by agarose gel analysis

PrincipleThe purines and pyrimidines in nucleic acid show absorption

maxima around 260nm (dATP: 259; dCTP: 272; dGTP: 253; dTTP: 247) of the DNA samples in pure without any contamination of protein or organic solvents. So absorption at 260nm can be taken to correctly assess the quantity. If the sample amount is less it can be visually estimated by gel electrophoresis.

Protocola) Take 3 ml of TE in two cuvettes (one for sample and the other

for reference) and calibrate the absorbance to zero at 260nm & 280nm both, after placing the cuvettes in the slots.

b) Replace 5µl of TE with the same amount of total DNA from the sample cuvette. Cover it with parafilm & mix properly by gently inverting the cuvette.

c) Wipe the outer walls of the cuvette with tissue paper and replace the cuvette in its slot. Record the absorbance at 260nm and 280nm.

d) Follow the same procedure for the other samples.e) One OD at 260nm corresponds to 50µg/ml of dsDNA, 33µg/ml

of ssDNA, 40µg of RNA and 20 - 30µg of nucleotides.f) Let the absorbance at 260nm be = A

Since 1.0 absorbance at 260nm corresponds to = 50.0µg/ml

Hence DNA concentration = 50 A dilution factor (µg/ml)

g) Judge the quality of DNA from the ratio of the OD values at 260nm and 280nm. Pure DNA has A260/A280 ratio of 1.8 and pure RNA of around 2.0. Protein with λmax of 280nm has an A260/A280 ratio of less than 1.0. Hence, a ratio of less than 1.8 suggests protein contamination and RNA contamination if it is equal or greater than 2.0

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10 - Restriction digestion of λ phage

DNA

IntroductionEndonucleases are enzymes that produce internal cuts, called

cleavage, in DNA molecules. Many endonucleases cleave DNA molecules at random sites, which have specific base sequence, such endonucleases are known as restriction endonucleses and the sites recognized by them are called recognition sites. The recognition sequences are different and specific for different restriction endonucleases or restriction enzymes.

Reaction Mixturea) Lamda DNA : 10µlb) Hind III : 2µlc) 10X reaction buffer : 2µld) Nuclease free water : 6µl

Final volume : 20µl

Protocola) Place the vial for reaction mixture on ice and add 2µl Hind III

enzymeb) Thaw the vials containing λ DNA (substrate) and 10X assay

buffer.c) Add 10µl of λ DNA in the vial.d) Add 2µl of 10X reaction buffer and 6µl of nuclease free water

in vial. Mix these tubes by vortexing.e) Incubate the vial at 37˚C for 1 hr.f) Prepare 1% Agarose gel for electrophoresis.g) After completing one hr of incubation and appropriate amount

of gel loading buffer in digested sample and load in Agarose gel.

h) Also load undigested substrate DNA and marker in gel for comparison.

i) Electrophorese the samples at 50-100V for 1-1.5 hrs.j) Visualize the DNA bands by UV Transilluminator.

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11 - Ligation of DNA fragments

IntroductionConstruction of any recombinant molecule of DNA is

dependent on ligation of 5’ phosphate and 3’ hydroxyl terminus. The ligase enzyme catalyses the formation of phosphodiester bonds between 5’ phosphate and 3’ hydroxyl terminus of dsDNA. The T4 DNA ligase has the unique ability to join sticky and blunt ends of DNA fragments.

Reaction Mixturea) Lambda DNA digest : 10µlb) T4 DNA ligase : 2µlc) 10X reaction buffer : 2µld) Nuclease free water : 6µl

Final volume : 20µl

Protocola) Place the vial for reaction mixture on ice.b) Take digested lambda DNA or digest it as earlier experiment

and add 10µl of it in vial.c) Thaw the vials containing T4 DNA ligase and assay buffer.d) Add 2µl of T4 DNA ligase and 2µl of assay buffer in vial.e) Add 6µl of nuclease free water in mixture and mix it by

vortexing.f) Incubate the reaction mix at 37˚C for 2 hrs in a pre set water

bath for ligation.g) Prepare 1% Agarose gel for electrophoresis.h) After completing 2 hrs of incubation and appropriate amount

of gel loading buffer in ligated sample and load in Agarose gel.

i) Also load digested substrate DNA marker in gel for comparison.

j) Electrophorese the samples at 50-100V for 1-1.5 hrs.k) Visualize the DNA bands by UV Transilluminator.

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12 - Polymerase Chain Reaction

IntroductionPCR (invented by Kary Mullis, 1980) is an in vitro method by

which specific DNA segments that lies between two regions of known sequences or primer binding sites are selectively amplified several folds in the presence of thermostable DNA polymerase. PCR is typically carried out using two oligonucleotide primers (one forward and one reverse) that flank the DNA fragments to be amplified. These primers hybridize with opposite strands of the target sequence and are oriented so that DNA synthesis by the polymerase proceeds across the region between the primers.

In general the amplification involves three basic steps, which are :(i) denaturation of template DNA in the presence of large molar

excess of each of dNTPs and primer.

(ii) annealing of the primer to the complimentary region on the template DNA and finally

(iii) extension of the annealed primers with thermostable DNA Polymerase.

As the extension products are complimentary to and capable of binding primers, successive cycles of amplification double the amount of target DNA synthesized in the previous cycles thus resulting in rapid exponential accumulation of the specific target DNA. The termini of this exponential reaction (dsDNA segment) are defined by the 5' termini of the primer and length by the distance between the primer binding sites. However, products of first cycle are heterogeneously sized, and the length may exceed distance between the primer binding sites. In second cycle DNAs of defined length are generated which are exponentially accumulated in late cycles of amplification and constitute the dominant product of PCR. Because longer molecules accumulate at a linear rate, they do not significantly contribute to the final mass of products.Since primer extension and annealing is carried out at elevated temperatures, the products become specific, the size and yield is also improved. Although extremely efficient under normal reaction conditions the amount of Taq DNA Polymerase becomes limiting factor after 30 cycles of amplification

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Reagents(i) Template DNA (ii) Primers (iii) dNTPs(iv) MgCl2 (v) 10X PCR buffer (vi) Taq DNA Polymerase (vii) Mineral oil (optional)

ProtocolDNA samples (12 ng) are amplified in 15µl reaction mixtures

containing 10X PCR buffer (10 mM Tris (pH = 9.0), 50 mM KCl, 1.7 mM MgCl2, 0.1 % Gelatin), 0.125 mM of each of the dNTPs, 9 ng primer and I unit of Taq DNA polymerase. A cocktail mix is prepared for required number of reactions plus one reaction to compensate the pipetting loss. This extra reaction mix is used as blank (without DNA) so as to check artifacts, if any.

a) For n no of reactions prepare a cocktail mix as follows(15µl): Buffer 1.5µl x (n+1); dNTPs 1.5µl x (n+1); MgCl2 0.12µl x (n+1); primer 1.8µl x (n+1); Taq DNA polymerase (3 units/µl) 0.3µl x (n+1); sterile DW 8.58µl x (n+1).

b) Arrange 0.5 ml thin walled flat cap PCR tubes in rack and label them for each genomic DNA.

c) Add 1.2µl (12 ng) of each genomic DNA to the corresponding PCR tubes arranged in the rack.

d) Add 13.8µl of the cocktail mix to each of the PCR tube containing genomic DNA.

e) Optional step if the thermal cycler is not equipped with heated lid: overlay a drop of mineral oil to the reaction mix to prevent evaporation of reaction mix at higher temperature during PCR cycles.

f) Close the caps and give a momentary spin.g) Place the tubes in the thermal cycler and set it to carry out

the following temperature profile: initial denaturing at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min & primer extension at 72°C for 2 min and finally primer extension at 72°C for 7 min & storing at 4°C.

h) Separate PCR products on 1.5% Agarose gel.i) Visualize and record the data with the help of Gel

Documentation system.

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13 - Elution of DNA from Agarose Gels

Introduction When a specific DNA of the same size is needed, then there is

need to isolate or elute the DNA from gel for further study. For example after PCR amplification of DNA you will find several bands in RAPD or specific bands along with some non specific bands with gene specific primers. In such cases you may require to elute the desired DNA fragments.

Protocol a) Excise the DNA fragment from the Agarose gel with a clear,

sharp scalpel. Weigh the gel slice and transfer it to a 1.5ml centrifuge tube.

b) Add 300µl of NE binding buffer to the centrifuge tube containing 100mg gel slice. Incubate at 50-60°C for 3-5 min and invert the tube occasionally until the Agarose gel is completely dissolved.

c) Transfer the above mixture to a miniprep spin column with a 2ml collection tube. Let it stand for 5 min. centrifuge at 13000rpm for 10-20 sec and discard the flow through.

d) Add 500µl of 80% isopropanol (or ethanol) to the spin column. Centrifuge at 13000rpm for 30 sec and discard the flow through.

e) Repeat the washing procedure in step d).f) Centrifuge at 13000rpm for an additional 1 minute to remove

the residual isopropanol.g) Place the spin column into a new 1.5ml microfuge tube. Let

the tube lid open for 2-3 min to volatilize isopropanol completely.

h) Add 50µl TE buffer or ultrapure water into the center part of the simax membrane in a spin column. Incubate at RT for 3-5 min. Centrifuge at 13000rpm for 1 min to elute DNA.

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i) Determine the quality of DNA fragments on 1% Agarose gel stained with EtBr. Store the purified DNA at 4°C for immediate use or at -20°C for future use.

14 - SDS-PAGE

Introduction Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis

(SDS-PAGE) is the most widely used method for quantitative analysis of protein samples. The separation of proteins is based on the size of the protein, hence this method can be used to determine the molecular weight of a given protein sample. SDS used is an anionic detergent. The samples to be run are boiled for 5 min in a sample buffer containing β mercaptoethanol, SDS and PMSF. β mercaptoethanol is used to reduce any disulphide bridges that are holding tertiary structure together. SDS denatures the protein and opens the protein into a rod shaped structure with a series of negatively charged SDS molecules along the polypeptide chain.one SDS molecule binds for every two amino acid residue. Sample buffer also contains an ionisable tracking dye, usually BPB, that allows the electrophoretic run to be monitored and sucrose or glycerol so that the sample can settle down in the well when loaded

Purpose of stacking gelStacking gel is used to concentrate the protein sample into

sharp band before it enters the resolving gel. This is achieved by utilizing the differences in ionic strength and pH between the electrphoretic buffer and the stacking gel, which involves a phenomenon known as istachophoresis. Stacking gel has larger pore size, which allows the protein samples to concentrate and move freely under the effect of electric field. Band sharpening is attained by the difference in the electrophoretic mobility of glycinate ions, protein-SDS complex and chloride ions in the loading buffer. For having a steady electric circuit all the species have to migrate at the same speed under the influence of the applied field.

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Field strength is inversely proportional to conductivity that is proportional to concentration.

Cl > Protein-SDS > GlycinateDue to lower concentration of protein-SDS complex they

concentrate in a very tight band between glycinate and chloride ion boundaries. Once glycinate ions reach the resolving gel due to higher pH environment they get easily ionized and their mobility increases.

Purpose of resolving gelOnce the protein-SDS complex enters the resolving gel, owing

to molecular sieving property of the gel, separation of the proteins in the sample is observed. Smaller proteins move more easily and hence move farther when compared to larger proteins. BPB being small molecule moves farther and forms electrophoretic front.

Sample preparation Samples for SDS-PAGE are prepared by adding 1X sample

buffer to the extracted protein samples. The samples are centrifuged and then the supernatant is transferred into an eppendorf tube. The samples are stored at -80˚C. Just before running, the samples are boiled for 5 min and placed on ice till loading.

Protocol a) Clean the glass plates before setting the gel mould.b) Use silicone as grease and apply on all spacers to be used.c) Apply spacers on their respective position.d) Seal the glass plates from three sides.e) First prepare resolving gel and pour in the gap between the

two plates.f) After the resolving gel polymerizes, prepare stacking gel and

pour it so that it layers on top of resolving gel.g) Place the comb immediately after pouring the stacking gel.h) Leave the setup for half an hour for the stacking gel to

polymerize and remove the comb upon polymerization of the gel.

i) Adjust the SDS-PAGE apparatus in such a way that it is parallel to the ground.

j) Remove the lower spacer, fix the gel to the apparatus using silicone grease, and tighten the screws.

k) Add 1X running buffer to the apparatus.l) Run the gel without loading sample for 5 -10 min.m)Prepare and load the samples and run at appropriate voltage

till the dye runs out.n) Remove the gel mould, transfer it into a tray with staining

solution, and leave overnight with intermittent rocking.

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o) Transfer the gel into the destaining solution till the bands appear properly.

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