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Nested LA PCR


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#1 Zach T

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Posted 14 March 2011 - 11:55 AM

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!

Edited by Zach T, 14 March 2011 - 11:56 AM.


#2 Trof

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Posted 15 March 2011 - 04:29 AM

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.

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#3 Zach T

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Posted 15 March 2011 - 02:38 PM

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?

#4 Zach T

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Posted 17 March 2011 - 09:22 AM

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