Normal PCR

Reference: Saiki et al., 1985, 1988

1. Prepare premix by combining
   10 x SuperTaq buffer
   dNTPs
   SuperTaq
   milli-Q water
   template (when common for all PCRs)
   primers (when common for all PCRs)
2. Aliquot premix into 0,5 ml eppendorf tubes
   Add template and/or primers (specific for each PCR)
   
   This final PCR mixture is composed of :
   10 mM Tris.HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl₂, 0.1 % (v/v) Triton X-100, 0.2 mM of each dNTP, 0.01 % (w/v) gelatine, 1 mM of each of the two primers that flank the region to be amplified, 0.01 ng template DNA, and 0.5 units of SuperTaq DNA polymerase (H.T. Biotechnology LTD, Cambridge, UK).
3. Optional (when working with volumes < 20 µl, or in a thermoblock without heating cap):
   Cover the reaction mixtures with light mineral oil
4. Place samples in a TRIO-Thermoblock (Biometra, Göttingen, Germany)
   Start program:
   Initial DNA denaturation 95°C 1 minute
   DNA denaturation 95°C 30 sec < -------|
   Primer annealing 50° to 60°C 30 sec | 25 x
   Primer extension 72°C 1 min/kb--------- |
   Final primer extension 72°C 5 min
   Storage 4°C infinite

Remarks:
An optimal annealing temperature can be determined with the computer program OLIGO ver. 2.0 of Rychlik and Rhoads, 1989.

When high efficiency and specificity of PCR amplification are required, 10% glycerol can be added to the reaction buffer (Lu and Nègre, 1993).

The yield of PCR product can be estimated by agarose gel electrophoresis. This also enables one to estimate the real number of DNA duplications during PCR (Fromant et al., 1995).

PCR products can be purified by phenol/chloroform/isoamyl alcohol extraction and Sephadex-G50 chromatography.