Ligation problems...

George Rutherford gruther at bilbo.bio.purdue.edu
Wed Dec 18 12:31:35 EST 1996


In article <Pine.ULT.3.91.961218114009.21727A-100000 at rowan>, SARAH LOUISE
RAWLINGS <slr2 at coventry.ac.uk> wrote:

> Could anyone please tell me why certain concentrations of ATP and the use 
> of Hexammine coblalt (II) Chloride in blunt ended ligations are 
> inhibitory to the ligation.
> I'm having terrible problems ligating a blunted vector and DNA insert 
> together!
> 

Sarah,

Although not directly on point, after years of fiddling with various
conditions for doing blunts, I have come to the conclusion that it really
doesn't much matter, so I do all my ligations the same way, using only the
usual buffer, ATP, ligase and DNA. I realize that this is heresy but  they
all work, although the blunts with lower efficiency. IMHO, the most
important thing in blunt ligations is ensuring that the ends are in fact
blunt. I generally use T4 Pol to polish the ends. I do know some people
who use PEG in their blunts, but I have an unreasoned paranoia about that
reagent and therefor eschew its use. Keep plugging and good luck.

George
> 
> 
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>         
>



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