The question says it all.
With normal PCR, since you don't have the methylated-DNA digestion step, you can simply stop after PCR or Restriction digest or ligation step. But with Q5 site directed mutagenesis, can I do the same? Like stopping after the PCR? Is it gonna affect the methylation state of the parental DNA, hence affect the digestion step?
Thank you very much
Hi,
You could stop after the PCR amplification and wait until the next day or the next week if you store your PCR product at 4°C or -20°C, respectively. I never faced any problems at this step. However, it might be more efficient to use the night for the DpnI treatment. Moreover, you could take a break prior to the transformation if you store the DNA properly. Nevertheless, it is always the best to do all steps at one day.
Good luck with your mutagenesis!
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