PrimerDigital

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 9-10°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 50°C up to 70°C).

PCR steps – the primers binding (usually from 55°C) and the polymerase extension (usually from 60°C to 72°C), we recommend to join into one step as 60°C. This step includes primer binding the target and polymerase extension at once; the recommended time for this step is 1 second for each 50 bases of PCR product. The denaturation of genomic DNA is easy with short step at 95-96°C, 5-20 seconds.

The PCR was performed in a 25 µl reaction mixture containing 25-50 ng DNA, 1x PCR buffer (with 2 mM MgCl₂ or MgSO₄), 0.2-1 µM of primer (for primer combinations – maximum 1 µM is total concentration), 0.2 mM dNTPs, 1 U Taq DNA polymerase and optimal additional 0.04U pfu DNA Polymerase (for long products amplification).

The PCR machine was programmed for amplification short and long (1,000-6,000 bases) amplicons:
1 cycle at 95°C 3 min, 25-30 cycles for 95°C (10-15 sec), 60°C (60 sec), 68°C (10-90 sec); and final extension step 68°C 5 min.
Amplification protocol for short (100-500 bases) amplicons:
1 cycle at 95°C 3 min, 25-30 cycles for 95°C (10 sec) and 60°C (5 sec); final extension step 68°C 2 min.

Amplification was performed in PTC-100 Programmable Thermal Controller (MJ research Inc., Bio-Rad Laboratories, USA) or a Mastercycler Gradient (Eppendorf AG, Germany) in 0.2 ml tubes or 96-well plates.

WebTools for general PCR and qPCR set-up.