help with pcr cloning
Lise Bonin
lbonin at fred
Wed Aug 16 18:07:54 EST 1995
To all pcr wizards:
I am trying to pcr a 3.0 kb fragment ouf of a plasmid. My primers are
30 mers with restriction sites on the ends for directional cloning if I
ever get that far. They also have GCGCGC (or CGCGCG ) "clamps" on the
ends. I have tried a variety of conditions such as MgCl2 concentrations
ranging from 1.5-5 mM and different cycling conditions with annealing
temps as low as 47 degrees (based on suggestions by Oligo 4.0 software)
and as high as 72 degrees. The basic cycling conditions I have tried
are: 94/5 min followed by 30 cycles of 94/1 min, 47/1 min, 72/2.5 min
followed by 10 min at 72 degrees. To date I have had no luck with this.
The only product I have seen with any of these conditions is an approx
800 bp fragment and that was weak. The reaction mix is as follows: 1x
pcr buffer (promega), 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 plasmid DNA,
linearized or not linearized. The reaction vol is 50 microliters. I
have run out of ideas. If you have any suggestions or magic solutions, I
would be most happy to hear them. Thanks!
More information about the Methods
mailing list