Cloning a PCR product

the End jgraham at bronze.ucs.indiana.edu
Tue Jun 28 17:23:57 EST 1994


I had an opportunity to estimate the amount of non-templated 3' A addition
by Taq in a recent project and found that only about half of the 
ends were so modified. You should be able to ligate "blunt-sticky"
into the vector without further modification.

Lately, I've felt that the most important aspect in recovering your
desired recombinant molecule will be the efficiency with which they 
transform your host. Try the Inoue method of peparing competent 
cells (growth at 18-22C, Gene 96 p. 23-28). I will routinely
generate a lawn of transformants from 1/10 of typical sticky-sticky
ligation with this procedure.

Jim
J. Graham
Indiana University Bloomington 






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