need help with cloning by PCR

Lise Bonin lbonin at fred
Wed Aug 16 17:28:15 EST 1995


To all pcr wizards:
I'm trying to pcr a 3 kb fragment from a plasmid - with no luck to date.  
My primers are 30-mers with 14 nt complementarity to the target 
sequence.  These primers also have GCGCGC (or CGCGCG) "clamps" on the 
ends as well as restriction sites for directional cloning if I ever get 
that far.  I've tried a variety of concentrations of MgCl2 (1.5-5 mM) and 
different cycling conditions.  So far, I've run the following conditions, 
varying the annealing temp from a low of 47 degrees (based on suggestion 
by Oligo 4.0 software) to 65 degrees or higher:  94/5 min, followed by 
94/1 min, 47/1 min, 72/2.5 min (25 to 30 cycles) and finally 72/10 min.  
The reaction mix is 1x taq pcr buffer(prom, 200 micromolar dNTPs, 25 
pmoles each primer, 1.5 - 5 mM MgCl2, 2.5 U Taq polymerase, 0.2 U Deep 
vent, and 200 ng linearized or unlinearized plasmid DNA.  Final reaction 
vol is 50 microliters.   The only improvement I see with the lower 
annealing temp is a weak product at approx 800 bp.  I've racked my brains 
(as well as my colleagues') and can think of no other solution.  I would 
most greatly appriciate any and all suggestions!  Thanks!

Lise
lbonin at fred.fhcrc.org



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