help with pcr cloning

Isaac Kim ikekim at merle.acns.nwu.edu
Tue Aug 29 17:00:02 EST 1995


In article <40ttoa$74n at mule.fhcrc.org>, lbonin at fred (Lise Bonin) says:
>
>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!

To my last response, I forgot to add one more thing. I think you may be 
using too musch template. In my experience, PCR's worked for template 
concentrations in the hundred picogram range. At higher template 
concentrations, some primers worked while others did not. 
Good luck.

Isaac.



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