Gel purification ligation problem

David Micklem dmicklem at cmgm.nospam.invalid
Wed Jul 18 08:51:14 EST 2001


[[ This message was both posted and mailed: see
   the "To," "Cc," and "Newsgroups" headers for details. ]]

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
>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

Hi Harry,

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.

HTH,

David

-- 
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
Unsolicited email will incur a US$100 processing charge.




More information about the Methods mailing list