problem in cloning
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:
> 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.
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?
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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