PCR Cloning

the End jgraham at bronze.ucs.indiana.edu
Tue Feb 22 09:41:45 EST 1994


There is a nice little literature out on this in Methods in Enzymology,
NAR, and Biotechniques that will get you further that any "kit".

A few options are to include restriction sites in the primer ends,
which worked well for me. As this is sometimes a problem, you can 
merely ligate your products into concatamers by treating them
with ligase and PEG and then digest the mutimeric product. No 
need to "polish" for this, as there are some which lack the 
3' addition in every batch and will ligate directly.

Optionally, you can take advantage of several techniques which
will incorporate fewer residues as your 5' primer ends, and allow
directional cloning. If you are cloning into a region of 
homology, you can digest the ends with ExoIII and create
sticky ends. In that case you may be able to transform your
annealed vector-insert without any ligation.

You may as you say "polish" the ends of your PCR product, or 
alternately which has worked well for us, add "T" s to a 
blunt vector digest by taking advantage of the same nucleotide
transferase activity of Taq that creates the problem. HOWEVER,
a note from NEB on EcoN1 suggests that ligating single base 
sticky ends is LESS eficient than blunt ends. This would
seem a problem with T-vectors, I would like to hear about
ligation efficincy with these if available.

Good luck. Try a search of the Science Citation Index for 93 for 
references, or I will post some if that is not possible.

Jim
J. Graham





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