Blunt end Ligation

micprf at lure.latrobe.edu.au micprf at lure.latrobe.edu.au
Wed Mar 23 04:37:27 EST 1994


In article <9403151925.AA13581 at ern.doe.ernet.in>, anjali at nii.ernet.in writes:
> Hi,
> My problem seems trivial but has been driving me nuts so any help from 
> anybody would be highly welcome. Let me explain what it is:
> I have a 11.2 kb plasmid which has a 1.8 kb luciferase gene as the reporter.
> I want to delete a 2.1 kb fragment from this plasmid (which is not a part
> of the reporter) which is released as a Xho1-Nde1 fragment. The resulting
> backbone of 9.1 kb was run on an agarose gel and purified. The ends were 
> end filled using Klenow(confirmed by adding radioactivity in the reaction)
> and then blunt end ligated. I got about 100-150 transformants (when transformed
> in DH5a). 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

Anjali,
Hard to tell what they are without more detailed info on restrition
sites etc. but one possibility is contaminants. Bear in mind if you
are electroporating and using good electrocompetent cells you need only
femtogram quantities of an intact contaminating plasmid to get 100
transformants. This level of contamination would never be seen in
an ethidium bromide stained gel and might even be hard to pick up
in a Southern blot with homologous probe. Cheers, Paul Fisher.




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