2 replies to this topic

[ashu2007]

Hi,

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.

[phage434]

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?

[bob1]

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.