Gel purification ligation problem

Bruno Cenni bruno.removethis.cenni at removethis.insel.ch
Thu Jul 19 01:26:22 EST 2001


...found the light box maker: http://www.clarechemical.com/



"Bruno Cenni" <bruno.removethis.cenni at removethis.insel.ch> wrote in message
news:3b567a86$1 at news.unibe.ch...
> 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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