Purifying clean plasmid vectors for ligation

Amie Franklin amief
Tue Mar 12 22:49:29 EST 1996


The variable in the reactions is probably the restriction enzymes. I never
digest a vector for subcloning overnight because contaminating nucleases can
chew off the overhangs (I digest 1-2 hr). Check your enzyme spec sheets and you
will probably see that there is a low but measurable nuclease contamination
level. An additional time-saving suggestion is to try a heat labile alkaline
phosphatase, you just heat kill the enzyme and then use the plasmid directly in
a ligation.

hope this is helpful,
Amie
amief at candelab.berkeley.edu




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