blunt end ligation problems

Wendy-Anne Smith wendy at ichr.uwa.edu.au
Wed Aug 21 20:25:26 EST 1996


In article <Morris_maduro-170896213737 at elegans.biology.ualberta.ca>,
Morris_maduro at biology.ualberta.ca (Morris Maduro) wrote:

> In article <ahouse-1208961639130001 at 129.64.32.172>,
> ahouse at hydra.rose.brandeis.edu (Jeremy C. Ahouse) wrote:
> 
> > Hello group,
> >    we are having a hard time with blunt end ligation. We are polishing
> > ends with pfu polymerase. Any suggestions given that? How sensitive are
> > ligases to salt concentration? I have read about the use of PEG and seen
> > reference to a protocol that changes the concentration during the ligation
> > reaction (first high conc favoring insert+vector, then low favoring
> > circularization). Any thoughts?
> > 
> >    Thanks,
> > 
> >    Jeremy C. Ahouse
> > 
Hi,

We are also trying to do the same thing i.e trying to clone Pfu generated
PCR products by blunt-end ligation with not to much success.  As for PEG
it works OK if you are cloning into a plasmid vector but it inhibits
transformation if you are using a phage vector like M13 (although it is
possible to remove the PEG by dialysis but that's fiddly).  Our ligations
did however work quite well though when we used the Promega high conc.
ligase as well as an vector:insert ratio of 1:3.

I hope this is helpful,
Wendy.

-- 
Dr. Wendy-Anne Smith

TVW Telethon Institute for Child Health Research
(Company Limited by Guarantee)
Western Australia

Fax 61 9 388 3414



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