Gel purification ligation problem

Dr. Harry Witchel Harry.Witchel at deletethis.bristol.ac.uk
Wed Jul 18 07:29:09 EST 2001


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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