cloning problem

Robert Whittier rfwhittier at hotmail.com
Mon Nov 18 03:47:26 EST 2002


nazarian80 at yahoo.com wrote:

>hi I have attempted to clone a 1.3 kb insert (Bamh1/Xho I) into a PRSETA 
>and PET28a >vector (Bamh1/xho1)  The vector has been gel purified and 
>fragments ligated (NEB T4 ligase >overnight at 14 C), and transformed into 
>BL21 DE3 cells . After plating/overnight incubation, very few colonies are 
>present OR not found, and >after screening for positives with Bamh1/Xho I, 
>there is no insert. Any suggestion/tips?

Try this:

You know those ligation kits containing PEG that often don't yield
such good results despite high ligation efficiency? The problem is
that they tend to produce linear multimers rather than recirculized
plasmids, and these just don't do you any good with a recA- strain.
But guess what. BL21 is recA+. It recircularizes the linear multimers,
so that ligations carried out in the presence of PEG give good
transformation efficiencies. At least it worked for me. Let us know
whether it solves your problem.

Bob Whittier

Amersham Biosciences K.K.
Tokyo






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