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Quick change mutagenesis No PCR product visible - (Jul/24/2012 )


Since last month I am trying to do a mutation by quick change mutagenesis, but I am unable to get the product. My primers are complementary to each other. I think because of that most of the time I am getting only one band which might be of those primer dimer.

Tm for those primers is 76.5, therefore I am using 71 as annealing temperature. I am using cloned pfu DNA polymerase AD.

My reaction mixture is of

Double distilled water: 35
pfu buffer: 5
25mM dNTP: 1.0
20uM primer 1: 1.0
20uM Primer 2: 1.0
Enhancer: 5.0
plasmid DNA: 1.0 (size of plasmid 6kb)
pfu AD: 1.0

and my conditions are
95C 5 minute
95C 1 minute
69.5/70.5/71.5 C 1 minute
72C: 6 minute

Go to Step 2 rep 8
95C 1 minute
71.5 1 minute
72C 6 minute
go to step 6 rep 14
72C 8 minute
hold at 4C

There is no problem in transformation because it is working for control plasmid, the same plasmid which I am using for quick change mutagenesis.

Please help me.


I'd recommend that you start by lowering your annealing temperature somewhere between 55-60. Do not worry about whether you can see a band, but simply transform your PCR product. Are you treating your PCR product with DpnI to get rid of template DNA?


To add to Phage's answer, the quikchange PCR is not an amplification reaction as such, more just a copying of the template, so you are not particularly likely to see a band on a gel.