Blunt end ligation debate.

Jarmo Niemi JANIEMI at FINABO.ABO.FI
Wed Jul 14 11:06:39 EST 1993


In <1993Jul14.085421.25969 at aristo.tau.ac.il> pc386 at ccsg.tau.ac.il writes:
> Following transformation, it was clearly evident that the CIP reaction failed
> to perform, as I obtained many colonies in the control plate of Blunted/CIPped
> self-ligated vector.
> 
> Now my debate: Should I "go for it" anyway, and try to pick up positive coloniesanyway, regardless of the fact that the CIP reaction failed, or should I insist
> on performing a more efficient CIP, and discard the colonies I already have.

I believe you should go for it. You only need one clone, right ?
If you make, say, ten minipreps of white colonies it only takes a day
and then most likely you will have at least one clone. If not, you only
have wasted a day.

It has been my impression that CIP not working is a sadly frequent 
problem, which is often not easy to solve. It appears that there 
still are bad batches of CIP from even the most reputable
manufacturers, so try another batch or another manufacturer. If
you find a good batch, HOARD it...

Had you not mentioned, that you have blue colonies in the control,
I would have warned you that in my experience of cloning abt. 300
bp fragments, I have encountered several clones, that are blue,
even if they have the correct insert. Don't trust Xgal.

Jarmo Niemi   Biochemistry, University of Turku, Finland, janiemi at finabo.abo.fi



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