blunt end ligation problems

Morris Maduro Morris_maduro at biology.ualberta.ca
Sat Aug 17 22:40:03 EST 1996


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
> 
> -- 
> Jeremy C. Ahouse, Biology - Brandeis University
> Waltham, MA 01432-1515
> 617.736-4954 (lab) 617.736-2405 (fax)
> ahouse at hydra.rose.brandeis.edu (email)


For cloning PCR products blunt-ended it can help (1) to use Pfu (as you
are), (2) clone into blunt-cut vector, like pBluescript cut with EcoRV, but
include some EcoRV in the ligation reaction (works as long as there are no
internal EcoRV sites of course).  Also, make sure that if the vector has
been DEphosphorylated, to ensure the primers in the PCR reaction ARE.  You
could probably make sure of this by adding T4 kinase and ATP before
purifying the product, or by kinase-treating the primers before PCR
(probably better). 

Maniatis et al. also suggests that the ATP concentration is important for
blunt-ended cloning.

Good luck,

Morris

-- 
Morris Maduro 
Dept of Biological Sciences 
University of Alberta
Edmonton, AB Canada 

Morris_maduro at biology.ualberta.ca



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