Does anybody know a good protocol for megaprimer-extension-PCR?
I am trying to mutate a gene on a expressionplasmid by generating megaprimers (about 1000 bp) with the Genemorph II mutagenesis Kit. I gel-purify those megaprimers and use them as primers for amplification of the whole plasmid in a second PCR round.
Unfortunately, I get only a very small amount of PCR-product in the second PCR-round (nearly no colonies on selective plates after transformation).
Does anybody have suggestions for improving the PCR-conditions for the mega-primer-extension PCR?
Thank you very much!
Dept of Molecular Cellular and Developmental Biology
University of Michigan
"Linda Foit" <[Only registered users see links. ]> wrote:
Except that what you are describing is *not* PCR. The amplification
is linear, not exponential - just like in "QuikChange" mutagenesis
Yes. We do this type of cloning all the time and it works quite well.
1. Use PfuFusion II polymerase in the second reaction at 65C extention
for 2 min/kbp in the presence of 5% DMSO, play with annealing
temperatures (we find that when megaprimer anneals to template over
very large length - like in random mutagenesis - no separate annealing
step is necessary).
2. Use lots of template. We use 50 ng/25 ul final reaction.
Make sure it is high quality. Lots of relaxed form results in
huge amounts of side reaction products of very high MW
products. We will be testing an idea of pre-treating
template with ligase before the reaction to see if repairing
nicks is helpful.
3. Use high efficiency electrocompetent cell. With these,
you can frequently get enough clones even when the overall
reaction worked very poorly. That means optimize voltage!
Cuvettes from different manufactures have slightly different
gap width, different strains have different optimal electric
filed intensity and even different batches made in the same lab
frequently vary in optimal voltages in 0.5-1 kV.
Suboptimal electroporation conditions can easily give 10X less