Gel purification ligation problem

Igor josstalin at home.com
Fri Jul 20 00:39:45 EST 2001


When you elute the gel-extracted fragment from resin use ligation 1X
buffer, then add linearized vector, ATP, ligase + extra required 10X
ligation buffer. 
It worked for me... 

"Dr. Harry Witchel" 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
> 
> --
> 
> 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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