Gel purification ligation problem

Chad Rappleye rappleye at biomail.ucsd.edu
Thu Jul 19 14:38:20 EST 2001


>From this thread it seems that everyone is pointing to UV-damage as the
most probable culprit for failed ligations.  To avoid UV altogether, I've
been using crystal violet to stain my gels as suggested in

"Crystal violet can be used to visualize DNA bands during gel
electrophoresis and to improve cloning efficiency", K.N. Rand, Technical
Tips Online 1996.

Using crytal violet, there is no UV at all.  You can sit and stare at
the gel all you want and trim it as small as you want.  The only
drawback is that it isn't as sensitive as Ethidium staining
but for cloning you should be using enough DNA to see it by crystal
violet anyway.

Chad Rappleye
Aroian Lab
UC, San Diego
(858) 822-1397

http://www.biology.ucsd.edu/labs/aroian

On 18 Jul 2001, Michael L. Sullivan wrote:

> 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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