Question about QuikChange II mutagensis method - Can it merge plasmids? (Sep/11/2007 )

Hello all,

I am trying to understand how the QuikChange II site directed mutagenesis method works. It seems that in the middle of the PCR reaction, the DNA would be linear after denaturing, and once the new strand is extended the nicked ends could come together and form a circular plasmid, but how do the two ends of one plasmid find each other, and not the matching end of a different plasmid (thus creating a larger plasmid containing two former plasmids repeated, with two sets of nicked ends)?

I'm not sure if the ends of the newly synthesised PCR'd plasmid come together until after transformation into the competent bacteria the kit uses. The original non-mutated plasmid is methylated and digested by Dpn I first to remove it. Try the kit manual for more details. You can probably download it from the Stratagene website.

Best wishes,
Ceri

Thanks for the reply Ceri, but still not quite sure I understand. Towards the end of the PCR reaction, wouldn't there be many copies of non-methylated plasmids annealed to each other as copies generated in previous cycles are used as templates in the current cycle? I thought that during the digestion with DpnI the original DNA would already be in a fairly low proportion. Am I missing something?