Unwanted blunt ending of sticky ends in cloning

micprf at lure.latrobe.edu.au micprf at lure.latrobe.edu.au
Thu May 13 04:11:27 EST 1993


Dear bionetters,
	I have been cloning using sticky end cutters AgeI and Sse8387I and
am finding high frequencies of clones in which the overhangs have been
removed at or before ligation. The result is that I am effectively getting
blunt end cloning which defeats the purpose I have in mind. The removal of the
overhangs happens either in the restriction digestion or in the ligation or
both and we are about to do some experiments to find out which. The buffer
I am using for both reactions is OnePhorAll Plus and the restriction
digestion is a simple double digestion. Has anyone else seen this? Is it likely
to be the ligase or one or both of the restriction enzymes? It effects both
the AgeI (5' overhang) and the Sse8387I (3' overhang) sites. Are any of the
three enzymes known to either have or be often contaminated with a single
strand nuclease activity? Please send responses to
fisher at lumi.latrobe.edu.au or micprf at lure.latrobe.edu.au.
				Thanks, Paul Fisher.



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