Hello, I have conducted a QuikChange site saturation mutagenesis with degenerate NNS primers to generate mutant libraries of certain residues of my enzyme. For all I have used annealing temperatures of Tm - 5. The reaction mixture is the PfuUltra II Hotstart PCR Master mix: https://www.chem-agilent.com/pdf/strata/600850.pdf
Only for half of my chosen residues could I observe plasmid DNA amplification.
Generally, even if DNA amplification was successful , after transforming competent E. coli cells with the respective plasmid, only very few colonies could be seen.
What are some problems, that can occur during the PCR cycles? I dont have any major hypotheses other that primer design might need to be reassessed.
hello,@
I would first lower the annealing temperature (Tm-10°C), then if you have clones but no mutant then check your dpn1 enzyme,
if you do not have transformants then make more cycles (for site directed mutagenesis you generally don't see the PCR product (13-16 cycles)) ...
you may also think to use another PCR enzyme (we shifted succecfully for the Q5 polymerase...
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