Cloning of gel-purified inserts

Frederik Wirtz-Peitz fwp at gmx.net
Sat Dec 16 13:36:22 EST 2000


Hello everybody,

This is a (rather lengthy) subcloning question:

I am relatively new to recombinant DNA and experiencing a lot of
difficulties subcloning gel-purified inserts. This is the case with both
restricted PCR products and restricted plasmids. I generally purify from
0.8-1.5% agarose gels using the Qiagen Qiaquick Gel Extraction Kit. The
purified inserts look fine when run out on a gel, and they concatamerize
nicely when self-ligated (as does the vector). I usually set up a series of
10µl ligations with molar insert:vector ratios somwhere between 10:1 and
0.3:1. Transformation of 1/5 of the reactions into TG1 recO and/or XL-10
results in 0-2 colonies (which I do not bother to screen since the no-insert
control is in the same range). A transformation control shows efficiencies
of around 1.e+6 cfu/µg of supercoiled plasmid DNA. For some reason I cannot
manage higher efficiencies despite using competent cells prepared by the
Hanahan method.

>From a theoretical perspective the ligations in questions should not be
considered problematic. The inserts range in size from 550 bp to 1.1 kb. The
two vectors used are 5.7 kb and 6.3 kb large. The inserts are flanked by
different, incompatible, restriction sites; XhoI and HindIII in one case,
SalI and BamHI in the other. Hence, I do not desphosphorylate the vector,
and the lack of colonies on the no-insert control shows that the vector has
been fully digested by both enzymes. I cannot know for sure whether the PCR
inserts have been fully digested as the sites are engineered into the
primers (with 4 bp tails), but the extensive laddering in self-ligation
suggests so.

The only constant in all these failed ligations is the insert having been
gel-purified. I have successfully subcloned PCR products that were not
gel-purified but simply restricted and subsequently cleaned up using a
Qiaquick PCR clean-up kit. I am therefore led to suspect there may be a
problem with gel purification of the insert. On the other hand, I do not see
how this can be rationalized in light of widespread use of gel purification
and also given the fact that even those successful ligations were still
based on gel-purified vector.

I am moderately desperate about this situation, and also running out of
time. If anybody has made similar experiences and/or can give general
advise, I would really appreciate that.

Thanks in advance,

Frederik

--
Frederik Wirtz-Peitz
Cambridge University
U.K.







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