Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

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!

-Zach T-

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?

-Zach T-


-Zach T-