help with pcr cloning

Isaac Kim ikekim at merle.acns.nwu.edu
Tue Aug 29 16:54:49 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!

I do routine PCR clonings in our laboratory. My feeling is that you just 
need to design new set of primers. Although your set of primers were 
designed with computer, the primers may not work. Other suggestions:
1) check the sequence of the primers. Are they synthesized correctly? 
Once, the company that I ordered primers from made a mistake and did not 
make the sequence as I have specified.
2) the product may be too long. Although normal PCR should work up to few 
kb, I have noticed that my PCR's do not go routinely for products greater 
than 1.5 kb. Try using long range PCR.
Good luck.

Isaac.



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