Cloning of gel-purified inserts

Tom Vink t.vink at med.uu.nl
Mon Dec 18 05:28:56 EST 2000


On Sat, 16 Dec 2000 18:36:22 -0000, "Frederik Wirtz-Peitz"
<fwp at gmx.net> wrote:

>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.
>
Probably a result of UV damage. Use high wavelenght UV (360nM) or
stain with crystal violet, this is much less sensitive but you donot
have UV damage. There is a small paper about this method in Tecnical
Tips Online on the Biomednet site . Hope this helps.
http://research.bmn.com/tto/search/record?uid=TTO.elstto00_01689525_4_t40022&node=TOC%40%40TTO%40001%40107%40001_107#tbl1


Greetings Tom






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