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Simple PCR to linearize plasmid not working

Phelan, Paul J. via methods%40net.bio.net (by Paul.Phelan from tufts.edu)
Wed Nov 16 20:24:19 EST 2016


Dear all,
I am trying to perform a simple PCR reaction to linearize a plasmid (so that I can insert a gene by Gibson assembly), but it is driving me mad by failing over and over again.  The plasmid is pcDNA3.1, and I have two PCR primers, designed to prime in opposite directions from my insertion site, so if it works I SHOULD get linearized plasmid!
But so far, if I use 25-30 cycles of amplification, I get several new bands of DNA on an agarose gel, both bigger and smaller than my template.  Far more bands than I expect.  But if I use less cycles, as in 18 cycles I get nothing.  The gel is just blank.  No PCR product whatsoever.  What I NEED TO see is a PCR product that migrates more slowly than my template (linearized plasmid), and nothing else.  But I either get far too many products of all sizes, or nothing.

Does anyone have any idea what I am doing wrong?  I double checked my primers and see nothing wrong with how they are designed.  I am at a loss.

Paul Phelan
Tufts University Boston


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