Subcloning PCR

Jeanne Loring jloring at genpharm.com
Thu Sep 1 19:12:21 EST 1994


                       Subject:                               Time:3:17 PM
  OFFICE MEMO          Subcloning PCR                         Date:9/1/94
Hello netters- I followed some of the recent thread about the problems with
cloning PCR fragments, and, since I now have the problem, I wish I'd listened
more carefully.  I cut my fragment with the restriction enzymes whose sequence I
believed I had included (with a 5'clamp), but there was no way to be sure they
both worked (SacI and XhoI).  Now I'm not getting inserts into Bluescript II.  A
colleague is having a similar problem with an entirely different sequence and
plasmid.  Was there a summary on the net about solutions?  I did read that
treatment of the Taq-produced fragment with the Klenow fragment of DNA
polymerase solved the problem for some people.  Has anyone else had luck with
that, or can you suggest another solution?
Thanks
Jeanne Loring
jloring at genpharm.com  




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