need help with cloning by PCR
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!
lbonin at fred.fhcrc.org
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