Cheapest PCR product cloning technique

Rich Dudley rdudley+ at pitt.edu
Fri Feb 19 08:48:25 EST 1999


> The simplest will be to use a proof-reading polymerase, which generates blunt-end
> products. Use primers which are not 5'-phosphorylated (to prevent concatenation of
> the PCR product). Clone the fragment directly into a blunt-end site of the vector. If
> you do the ligation in the presence of the enzyme which which you opened the vector,
> you will prevent re-circularization of the vector.

One caveat here--if your unknown sequence contains the enzyme site you are incubating
with, you won't have any of those clones either.

rich

--- --- --- -- -- -- --- --- ---
Richard J. Dudley (rdudley+ at pitt.edu)
Research Specialist V
Dept. of Cell Biology and Physiology
University of Pittsburgh
http://www.cbp.pitt.edu
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