cloning PCR products

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Thu Sep 29 13:36:38 EST 1994


In response to the thread about having trouble cloning PCR products
with r. sites incorporated in the primers.

Many people fall into the trap of getting intimidated about the 
difficulty of cutting these sites and then dump in so much r. enz.
that the inevitable contaminating activities in them destroy the
ends.  eg. If you put 1 ul (20U) of enzyme in with x ul PCR product
(say 0.5 ug) and then decide to digest overnight (say 15 hr.), you
just did a 600 x overdigestion.  The enzymes are generally only
tested by the manufacturer out to about 100 x overdigestion.
The problem is inherently irreproducible; getting away with it
in the past doesn't mean it'll work again in the future.

Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




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