Hello. The laboratory where I work has received a pFL Multibac construct from another lab. This construct contains apart from the vector backbone, four of the genes that we are interested. However, we need to perform specific point mutations in one of the genes that we are interested. Therefore, we thought of performing a PCR mediated mutagenesis. However, we have some doubts if this procedure is the most suitable because of the huge size of the plasmid (16570bp).
Thank you for your help.
I've used the Pfu based polymerase 'Phusion' (proofreading/exonuclease containing, essential for generating fragments with a low error rate) for generating mutants in a 12kb construct without issue so, decreased efficiency aside, I don't think there should be any other intrinsic issues with a larger fragment. It is very important that the template DNA prep is as clean as possible so as not to introduce further sources of inefficiency.
Stabilising the DNA/polymerase complex with increased Mg2+ (2 - 3.5 mM) is important but it's a balance between yield and specificity.
As with any PCR, dependant on the vagaries of the template, some enzymes work better than others so be prepared to try a few out (e.g. Pfu turbo) polymerases.
Good luck!!
I agree with Marcus. I do not think the size of your plasmid will be a problem. I have used the QuickChange Site-directed mutagenesis kit several times. It works well. Do not hesitate to try that for your experiment. Good Luck!!!
I have never tried mutagenesis with that huge plasmid, but be careful not introduce unwanted mutations. If it is 17kB you can try to replace region of your interest with mutation in your plasmid either using traditional cloning if restriction sites permit or kit based recombination. I have used kit based recombination to clone in a 2 kB fragment in to 38kB adeno vector and had worked
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