Blunt end Ligation

anjali at nii.ernet.in anjali at nii.ernet.in
Wed Mar 16 04:25:06 EST 1994


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



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