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 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 (T_m^min), our empirical formulae:

Ta = T_m^min + ln(s),

where s 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 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 98°C 30-60 sec; 25-32 cycles for 98°C (5-10 sec), 68-72°C (30-90 sec); and final extension step 72°C 5 min.

Amplification protocol for short (100-1,000 bases) amplicons:
1 cycle at 98°C 30-60 sec; 25-32 cycles for 98°C (5-10 sec) and 64-72°C (1-30 sec); final extension step 72°C 5 min.

PCR reaction setup calculators

PCR and qPCR reaction setup calculator; tool for planning PCR and qPCR reactions, mixing solutions.

Note: If you don't see the Java Web Start application's button, Java application needs the Java Runtime Environment (JRE) or complete Java Development Kit (JDK) (Sun), can be downloaded from java.com), or you might need to enable the JavaScript interpreter in your browser so that the Deployment Toolkit script can function properly.

Reference apply to all Web Tools update, if you use it in your work please cite:

Kalendar R, Lee D, Schulman AH 2011. Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics, 98(2).
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