Gel purification ligation problem

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Wed Jul 18 09:34:48 EST 2001


As somebody else suggested already, one frequent culprit is
over-irradiation on a short-wave UV light box.  I find that using a hand
held longwave (365 nm) UV lamp works very well for isolating fragments.  I
use it underneath the gel tray to transilluminate the gel.  Even relatively
long exposures to the lamp (several minutes) don't seem to effect the
"ligatability" of fragments in my hands.

Hope this is useful.

Mike

>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

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

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