Inclusion of restriction sites in PCR primers

Alex Merz merza at ohsu.edu
Sat Nov 2 23:58:04 EST 1996


In article <55a1sc$56r at bignews.shef.ac.uk>,
Kevin Mulcahy  <K.Mulcahy at sheffield.ac.uk> wrote:

I've had good luck doing exactly what you're trying, including the 
use of MacVector as a primer design aid. The only things I'd 
suggest are:
o Make sure that you choose primers that are GC-rich in the 3' end;
  the inclusion of this "clamp" probably reduces end-flutter and
  improves priming efficiency

o Be certain that the added sequences do not introduce secondary
  structure or anneal to the reverse primers

o Some polymerases work a _lot_ better in our hands than others. If
  only Stratagene could reliably ship Pfu... sigh.

-Alexey
 




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