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Polymerase Chain Reaction (PCR)
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Procedure:

 

The protocol describes how to amplify a segment of double-stranded DNA in a chain reaction catalyzed by a thermostable DNA polymerase. It is the foundation for all subsequent variations of the polymerase chain reaction.

 

Materials

 

Buffers and Solutions

 

10x Amplification buffer
Chloroform
dNTP solution (20 mM) containing all four dNTPs (pH 8.0)

 

Enzymes and Buffers

 

Thermostable DNA polymerase
Nucleic Acids and Oligonucleotides
Forward primer (20 μM) in H2O
Reverse primer (20 μM) in H2O
Template DNA.

 

Dissolve template DNA in 10 mM Tris-Cl (pH 7.6) containing a low concentration of EDTA (<0.1 mM) at the following concentrations: mammalian genomic DNA, 100 μg/ml; yeast genomic DNA, 1 μg/ml; bacterial genomic DNA, 0.1 μg/ml; and plasmid DNA, 1-5 ng/ml.

 

Method

 

  1. In a sterile 0.5-ml microfuge tube, mix in the following order:
  2.  

     

    REAGENTS AMOUNT(μl)
    Deionized water 37.5 μl
    Taq assay buffer(10x) 5 μl
    Template DNA 1μl
    dNTPs mix 2 μl
    Forward primer 2 μl
    Reverse primer 2 μl
    Taq DNA polymerase 5 μl

     

    The table below provides standard reaction conditions for PCR. Mg2+ (1.5 mM) ;KCl(50 mM) ;dNTPs (200 μM) ;Primers(1 μM );DNA polymerase (1-5 units); Template DNA(1 pg to 1 μg ).

     

    The amount of template DNA required varies according to the complexity of its sequence. In the case of mammalian DNA, up to 1.0 μg is used per reaction. Typical amounts of yeast, bacterial, and plasmid DNAs used per reaction are 10 ng, 1 ng, and 10 pg, respectively.

     

  3.  If the thermal cycler is not fitted with a heated lid, overlay the reaction mixtures with 1 drop (approx. 50 μl) of light mineral oil. Alternatively, place a bead of wax into the tube if using a hot start protocol. Place the tubes or the micro titer plate in the thermal cycler.
  4.  

  5. Amplify the nucleic acids using the denaturation, annealing, and polymerization times and temperatures listed below.
  6.  

  7.  Withdraw a sample (5-10 μl) from the test reaction mixture and the four control reactions, analyze them by electrophoresis through an agarose gel, and stain the gel with ethidium bromide or SYBR Gold to visualize the DNA.
  8.  

    A successful amplification reaction should yield a readily visible DNA fragment of the expected size. The identity of the band can be confirmed by DNA sequencing, Southern hybridization and/or restriction mapping. If all is well, lanes of the gel containing samples of the two positive controls (Tubes 1& 2) and the template DNA under test should contain a prominent band of DNA of the appropriate molecular weight. This band should be absent from the lanes containing samples of the negative controls (Tubes 3 & 4).

     

  9.  If mineral oil was used to overlay the reaction (Step 2), remove the oil from the sample by extraction with 150 μl of chloroform. The aqueous phase, which contains the amplified DNA, will form a micelle near the meniscus. The micelle can be transferred to a fresh tube with an automatic micropipette.
  10.  

    Do not attempt chloroform extractions in micro titer plates. The plastic used in these plates is not resistant to organic solvents.

 

Recipes

 

Amplification Buffer:

 

500 mM KCl.
100 mM Tris-Cl (pH 8.3 at room temperature).
15 mM MgCl2.
Autoclave the 10x buffer for 10 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. Divide the sterile buffer into aliquots and store them at -20oC.

 

KCl

 

Dissolve an appropriate amount of solid KCl in H2O, autoclave for 20 minutes on liquid cycle and store at room temperature. Ideally, this 4 M solution should be divided into small (approx. 100 μl) aliquots in sterile tubes and each aliquot thereafter used one time.

 

Tris-Cl

 

Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH to the desired value by adding concentrated HCl.

 

pH HCl
7.4 70 ml
7.6 60 ml
8.0 42 ml

 

(1 M) Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the solution to 1 litre with H2O. Dispense into aliquots and sterilize by autoclaving. If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris solutions is temperature-dependent and decreases approx. 0.03 pH units for each 1oC increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at 5oC, 25oC, and 37oC, respectively.

 

dNTP Solution

 

Dissolve each dNTP (deoxyribonucleoside triphosphates) in H2O at an approximate concentration of 100 mM. Use 0.05 M Tris base and a micropipette to adjust the pH of each of the solutions to 7.0 (use pH paper to check the pH). Dilute an aliquot of the neutralized dNTP appropriately, and read the optical density at the wavelengths given in the table below. Calculate the actual concentration of each dNTP. Dilute the solutions with H2O to a final concentration of 50 mM dNTP. Store each separately at 70oC in small aliquots. For polymerase chain reactions (PCRs), adjust the dNTP solution to pH 8.0 with 2 N NaOH. Commercially available solutions of PCR-grade dNTPs require no adjustment.

 

 

 

Base wavelength(nm) Extinction Coefficient(E) (M-1cm-1)
A 259 1.54 x 104
G 253 1.37 x 104
C 271 9.10 x 103
T 267 9.60 x 103

 

For a cuvette with a path length of 1 cm, absorbance = EM. 100 mM stock solutions of each dNTP are commercially available .

 

Precautions

 

Chloroform

 

Chloroform CHCl3 is irritating to the skin, eyes, mucous membranes, and respiratory tract. It is a carcinogen and may damage the liver and kidneys. It is also volatile.  Avoid breathing the vapours.  Wear appropriate gloves and safety glasses. Always wear a chemical fume hood.
 

 

 

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