Blunt end Ligation

Marinella Callow marinel at iserver.onyx-pharm.com
Thu Mar 17 21:23:31 EST 1994


In article <9403151925.AA13581 at ern.doe.ernet.in>, anjali at nii.ernet.in
wrote:

 On doing a colony hyb. with luciferase only 7 transformants picked
> up. These transformants did not pick with the deleted 2.1 kb fragment.
> What are the rest of the transformants? 
> The restriction pattern of these "positives" is absolutely weird. I'm not
> even getting the right size on linearization which should be 9.1 kb instead
> it is giving a band in the 5-6 kb region.
> Can anybody tell me what is the problem? And also suggest me modified 
> protocols which should be followed at any step?
> Thanks,
> Anjali.
> anjali at nii.ernet.in

I have pondered the question often "what are these smaller plasmids?". Well
recently someone suggested that this occurs more often when you are using
gel purified vector...which of course has been exposed to EtBr and UV. In
future you might just cut the vector with the appropriate enzymes to delete
the fragment and do a mixed ligation. Your deleted plasmid will be
tranformed at a higher frequency than the undeleted plasmid. Make sure that
your digest is as complete as possible. DO Nde1 first since this has a very
short half-life and moniter the digest,



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