PCR – basic mtDNA and nDNA


1. 7.6 μl ddH2O
2. 1.2 μl 25mM MgCl2
3. 1.2 μl 10mM dNTPs
4. 1.5 μl 10x (NH4)2SO4 PCR Buffer (no MgCl2)
5. 1.5 μl 2μM primer1
6. 1.5 μl 2μM primer2
7. 0.3 μl of 1 U Taq polymerase

Mix and add 14 μl of master mix to each reaction. Add 1 μl DNA (5-50 ng/μl).


1. 68°C for 60 sec. (prewarm)
2. 93°C for 5 sec. (denature)
3. 48-55°C for 35 sec. (anneal)
4. 68°C for 90 sec. (extend)
5. 68°C for 7 min. (final extend)


Repeat the parameters 2 – 4 35 times. I have found that a 2 or 5 minute “hot start” at 94°C does not work well – it depurinates DNA and degrades Taq. The last 68°C extension can also be left out. This is the standard mix for PCR reactions which works well and normally results in concentrated enough PCR product that be purified directly. If the PCR product is not strong enough or there are multiple bands, several solutions exist. These include lowering annealing temperature, increasing MgCl2 concentration, performing a touch-down or touch-up PCR, redesigning primers, cutting out and reamplifying the PCR band, or doing a nested PCR. In the case of multiple PCR bands specifically, the use of internal sequencing primers can give clean sequences.

 

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