ligation of incompatible sticky ends ???

colossus... s535290 at aix1.uottawa.ca
Fri Jun 13 19:19:27 EST 1997


Here's a little stumper for you fine folks...

The expression vector I'm cloning into will only accept NcoI/HindIII
inserts. It already carries a reporter gene, which you can excise by
NcoI/HindIII double digestion. I subsequently run such a digest, cut the
vector band out (9 Kb - the excised reporter is 1.7 Kb) and gel purify it.

Ligating into this doubly cut vector is being a pain in the ass on its
own, but when I do a self ligation control, I manage to get a few
colonies. I suppose there is a possibility of carryover of uncut vector
during electrophoresis and gel purification...is this what's happening ? I
always thought I was rather careful when doing gel purification of
fragments and this is unnerving to say the least !!!

comments ?

TIA,

Ed




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