help with pcr cloning

[Sailesh Surapureddi]

To: methods-and-reagents at net.bio.net
From: lbonin%fred at hulio.liu.se (Lise Bonin)
Subject: help with pcr cloning
Date: 16 Aug 1995 23:07:54 GMT

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!