Overlap extension PCR

Louis Hom lhom at OCF.Berkeley.EDU
Thu Sep 9 12:57:52 EST 2004

FWIW, when I have trouble with overlap extension, I try using super-high 
concentrations of the two fragments, either by purifying them together on 
the same Qiagen column or by EtOH ppn.  So, I typically end up with 
probably 500-1000 ng of each fragment in a 50 ul PCR reaction (no 
primers).  I usually cycle for 10 or 20 rounds, then take 2-5 ul of that 
reaction and use it as template in a fresh reaction (1 or 2 x 50 ul) with 
the outside primers.  It may seem like a black box or handwaving, but it 
usually works if I'm having trouble otherwise.
Lou Hom >K'93			     
lhom at ocf.berkeley.edu		

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