Gel purification ligation problem

Bruno Cenni bruno.removethis.cenni at removethis.insel.ch
Thu Jul 19 01:13:26 EST 2001


Hi Harry,

as Wolfgang suggests, don't even bother to purify the band. Use low melt
agarose (eg Promega's or other that won't inhibit ligase) either cut out the
band as small as possible (or alt. cut a trough into the gel just ahead of
the band, remove the TAE just to the level of the upper edge of the gel,
fill the trough with TAE, run the band into the trough and collect it). We
use the first method routinely (cut eg. 1 ug DNA, use 1ul agarose of vector
band and molar ratios of the insert bands). The crucial step is usually the
transfomation (needs to be better 10E8/ug). For the UV box thing: a plain
blue light box lights up EtBr, you then need orange filter to see it (blue
and orange plexiglass/plastic). There's one commercially available, can't
remember the company though.

cheers

Bruno


"Dr. Harry Witchel" <Harry.Witchel at deletethis.bristol.ac.uk> wrote in
message news:3B558261.8F6053E9 at deletethis.bristol.ac.uk...
> Hi folks --
> In my lab we are currently having problems with gel
> purification of DNA from agarose.  We are using Qiagen gel purification
> kits with all of the extra steps, and we are getting good yields of
> fragments (as judged by running an aliquot of the yield on a gel), but
> absolutely everything we purify is unligatable (as judged by failed
> ligation-transformations).  We know the restriction enzymes (and ligase
> and competent bugs) are working because if we do not gel purify, we can
> get
> the vectors to circularise (ie non-gel purified vector + no ligase -->
> 14 colonies, while non-gel purified vector + ligase --> 200 colonies).
> We suspect there may be something coming out of our gel purification
> step that contaminates ligation; we currently purify from Biorad
> molecular biology grade normal melting point agarose.  Do you have any
> suggestions or ways to move forward on the gel purification front?  When
> you reply, please note that you have to delete "deletethis" from my
> email address.
> All the best,
> Harry
>
> --
>
> Dr. Harry J. Witchel
> Dept. Physiology
> Medical School
> Bristol  BS8 1TD
>    England
> 44-(0)177-928 7817
> Harry.Witchel at deletethis.Bristol.ac.uk
> To send me an email, use the above address, but delete "deletethis."
> from the address
> http://www.bris.ac.uk/Depts/Physiology/Staff/HW/hw.htm





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