Gel purification ligation problem
dmicklem at cmgm.nospam.invalid
Wed Jul 18 08:51:14 EST 2001
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In article <3B5582C8.23CAAD04 at bristol.ac.uk>, Dr. Harry Witchel
<Harry.Witchel at deletethis.bristol.ac.uk> wrote:
>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
>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
> All the best,
You aren't by any chance using a short-wave UV gel box to cut out your
bands are you? 254nm UV (and even mid-wave (315nm??, I forget)) messes
up ligation/transformation efficiency _really_ fast.
D.R. Micklem, Time flies like an arrow...
Dept. of Anatomy, Fruit flies like a banana.
Cambridge University, Email:dmicklem at cmgm.stanford.edu
Cambridge, UK Phone: +44 (1223) 333776
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