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

Plasmid DNA: 1–5 ng/mL

Method

In a sterile 0.5 mL microfuge tube, mix in the following order:

Reagents Amount (µL)

Deionized water 37.5 µL Taq assay buffer (10×) 5 µL Template DNA 1 µL dNTP mix 2 µL Forward primer 2 µL Reverse primer 2 µL Taq DNA polymerase 5 µL

The values provided are standard reaction conditions for PCR:

Mg²⁺: 1.5 mM

KCl: 50 mM

dNTPs: 200 µM

Primers: 1 µM each

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.

For mammalian DNA, up to 1.0 µg is used per reaction.

Typical amounts for yeast, bacterial, and plasmid DNAs per reaction are 10 ng, 1 ng, and 10 pg, respectively.

If the thermal cycler is not fitted with a heated lid, overlay the reaction mixtures with one drop (~50 µL) of light mineral oil. Alternatively, place a bead of wax into the tube if using a hot start protocol. Then, place the tubes or the microtiter plate into the thermal cycler.

Amplify the nucleic acids using the recommended denaturation, annealing, and polymerization times and temperatures.

After amplification, withdraw a sample (5–10 µL) from the test reaction mixture and the control reactions. Analyze them by electrophoresis through an agarose gel and stain the gel with ethidium bromide or SYBR Gold to visualize the DNA.

A successful amplification should yield a readily visible DNA fragment of the expected size. The identity of the band can be confirmed by DNA sequencing, Southern hybridization, or restriction mapping.

If the reaction is successful:

Lanes containing the two positive controls (Tubes 1 & 2) and the test sample should show a prominent band of the appropriate molecular weight.

No band should appear in the lanes containing the negative controls (Tubes 3 & 4).

If mineral oil was used to overlay the reaction, remove the oil by extraction with 150 µL of chloroform. The aqueous phase containing the amplified DNA will form a micelle near the meniscus, which can be carefully transferred to a fresh tube using a micropipette.

Important: Do not attempt chloroform extractions in microtiter plates — the plastic 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 the solution for 20 minutes using the liquid cycle and store it at room temperature. Ideally, the 4 M KCl solution should be divided into small aliquots (approximately 100 µL each) in sterile tubes. Each aliquot should be used only once to maintain sterility.

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.