PCR amplification protocol

The higher quality of primers is help to save PCR efficiency at changing PCR conditions. PCR reaction can set up in room temperature and performed without hot-start enzymes.

The range of optimal annealing temperature (Ta) was calculated Tm of primer or optionally plus 6-12°C, and in practice PCR efficiency was tested with gradient annealing temperature using MasterCycler Gradient (Eppendorf). For primer combinations with very different Tm, the optimal annealing temperature was chosen according to lowest Tm primer (primer with CG content higher then 50% is tolerant to wide annealing Ta, from 55°C up to 72°C).
The optimal annealing temperature for PCR is calculated directly as the value for the primer with the lowest Tm (Tmmin), our empirical formulae:

Ta calculation
where L is length of PCR fragment.

PCR steps - the primers binding (usually from 55-60°C) and the polymerase extension (usually from 50°C to 72°C), we recommend to join into one step as 68-72°C. This step includes primer binding the target and polymerase extension at once; the recommended time for this step is 1 second for each 100 bases of PCR product. The denaturation of genomic DNA is easy with short step at 98°C, 5-10 seconds.

The PCR performed in a 25 µl reaction mixture containing 25 ng DNA, 1x ThermoPol® buffer (with 2 mM MgCl2 or MgSO4), 20-300 nM of primer (for multiplex PCR, primer combinations – maximum 1 µM is total concentration), 0.2 mM dNTPs, 1 U Taq DNA polymerase and (optionally) additional 0.01U Pfu DNA Polymerase (for long products amplification).
A polymerases mix consisting of 100-500 units of Taq DNA polymerase with 1 unit of Pfu DNA Polymerase greatly increased efficiently of amplification for long bands and the accuracy of the PCR.

ThermoPol® Buffer, 1X: 20 mM Tris-HCl (pH 8.8, 25°C), 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton® X-100.

The PCR machine was programmed for amplification short and long (2,000-6,000 bases) amplicons:
1 cycle at 95°C 90 sec; 25-32 cycles for 95°C (15 sec), 68-72°C (60-400 sec); and final extension step 72°C 5 min.

Amplification protocol for short (100-2,000 bases) amplicons:
1 cycle at 95°C 90 sec; 25-30 cycles for 95°C (15 sec) and 64-72°C (60 sec); final extension step 72°C 5 min.

Touchdown amplification protocol for short (100-2,000 bases) amplicons:
1 cycle at 95°C 90 sec; 10 cycles for 95°C (15 sec) and 72°C (10-60 sec); 20-25 cycles for 95°C (15 sec) and 55-65°C (10 sec) and 72°C (10-60 sec); final extension step 72°C 5 min.

PCR reaction setup calculators