In article <9403151925.AA13581 at ern.doe.ernet.in>, anjali at nii.ernet.in writes:
> 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?
> Anjali.
Hi,
I don't know what your problem is, but when I do this kind of deletion I always
fill the ends before running on a gel (Klenow is very sensitive to agarose
contaminants) - just add dNTPs and Klenow for 20 minutes before you run your
reaction products. It works much better, and I wouldn't bother with the
isotope. The other modification you ought to try is to heat kill the ligase
and treat the ligation mix with XhoI or NdeI, or both, before transforming. I
usually make the ligation up to 100ul with RE buffer, cut, then transform about
10% of it. Alternatively, just add one of the restriction enzymes into your
ligation. This way you ought to get 100% genuine deletion derivatives.
--
Cheers,
Martin
NNNN NN Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz) ZZZZZZZ
NN NN NN Cytogenetic and Molecular Oncology Unit ZZZ
NN NN NN Christchurch School of Medicine ZZZ
NN NNNN Christchurch, New Zealand ZZZZZZZ
Phone (64-3)364-0880 Fax (64-3)364-0750
