problem in cloning

David Micklem dmicklem at cmgm.nospam.invalid
Tue Feb 27 11:31:39 EST 2001


In article <002b01c0a095$fb65d4e0$8c01a8c0 at tn.nic.in>, R. Jayakumar
<jakku at mrna.tn.nic.in> wrote:

>Dear friends
>    I need to clone a BamHI/BglII insert of 4 kb in size into the BamHI site
>of pAT19 which is a 6.6kb vector contaiing the rk2 origin for triparental
>mating.  I am facing problem with ligation, even though I am using fresh
>ligase from GIBCO BRL.  I am not getting any blue or white colonies on
>erythromycin (150ug/ml) plates with IPTG/X-Gal. ACtually there are no
>colonies at all growing, showing that there may not even be any
>self-ligation itself.  I have tested the viability of the cells and they are
>OK.  Competency is also not a problem.
>   the insert was eluted from gel using the BM's agarose gel extraction kit.
>Could this be the problem?  Please help me out.
>thank you for any useful suggestion.
>sincerely
>jayakumar

What do your controls look like? (eg uncut vector). I don't suppose you
could have fallen for the classic mistake - cutting your band out on a
short-wave UV transilluminator?

David

-- 
D.R. Micklem,              Time flies like an arrow... 
Dept. of Anatomy,          Fruit flies like a banana.
Cambridge University,      Email:dmicklem at cmgm.stanford.edu 
Cambridge, UK              Phone: +44 (1223) 333776
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