Nested LA PCR - Any success? (Mar/14/2011 )

I've been having troubles getting good amplification of my long and accurate PCRs (LAPCR). I've been using two different kits, Takara and Bio-rad's iProof. I've been most successful with Takara but haven't gotten very reproducible results. I'm trying to amplify targets ranging from 13kb to 23kb. Part of my issue I believe was I was vortexing the template (very briefly) but this probably caused mechanical shearing and reduced the amplifiable amount of DNA.

I just got new template so I'm going to retry these rxns but I was wondering if anyone has had better (or just even any) success running a nested pcr version of lapcr. And if so, what was your protocol and profile? Thanks!

Yes, don't vortex. Check also the method of DNA isoltion. There could be more shearing using column kits (check the manual), than using the old phenol/chlorophorm method.

Thanks. Yeah i've stopped vortexing the DNA but still haven't gotten any amplfication. Going to try nesting it next time.

Anyone tried nesting a lapcr before?

bump