Polymerase chain reaction (PCR)
PCR (Polymerase chain reaction) are typically carried out in 25 ml reactions. For standard PCRs, such as used for mapping, DNA is amplified by use of Taq DNA polymerase (Roche).
If a specific product is required, such as was used when cloning, then a proof reading polymerase such as pwo or Expand-HiFidelity (Roche) should be used. Typically a PCR mixture constituted 1.25 Units Polymerase, 0.4 mM Fwd and Rev primers, 200 mM dNTPs (dATP, dCTP, dGTP, dTTP), and 1xPCR buffer (10x PCR buffer: 100mM Tris.Cl pH 8.3, 500mM KCl, 15mM MgCl 2, 0.1% gelatin, 0.5% tween ® 20, 250 m g/ml BSA).
Polymerase chain reaction (PCR) amplification consists of an initial two minute denaturing step at 96°C. Ffollowed by a cycle of denaturing, annealing and extension conditions. The number of cycles and the temperatures of each step is variable. A typical PCR has 30 cycles of (i) 30 secs denaturing at 94°C (ii) 30 secs annealing at 62°C (iii) 2 minute extension at 72°C. This cycling is followed by a 10 minute final extension. The main changes in PCR conditions involves varying the number of cycles performed and the use of different annealing temperatures.