Gel purification ligation problem

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Wed Jul 18 08:21:55 EST 2001


Harry, 

recently I read in Maniatis about a very simple ligation 
procedure: They suggest just to cut out the slice from low 
melt agarose and melt it at 60 degC. Then mix aliquots of 
vector and insert without further purification into ligation mix 
before it re-solidifies and ligate.

Could there be a DNAse in one of your solutions?

HTH
Wolfgang

> 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
> 
> 


[tiss mezzage wahs broduceRd using TYPO GENERATOR zoffwer]
-----
Dr. Wolfgang Schechinger
Lab N233 (c/o Dr. Steve Roffler)
Institute of Biomedical Sciences
Academia Sinica
128 Yen-Chio Yuan Rd. Sec.2
Taipei 115
Taiwan R.O.C.
Tel +886-2-2789-9152
Fax +886-2-2782-9142
Mobile +886-925-136893
e mail wolfsc at ibms dot sinica dot edu dot tw
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