Rapid Ligation

[Gary K.]

In article <U10784.14.00098F9A at UICVM>, U10784 at UICVM (Victor Levenson) wrote:

> >In article <4fgj3k$4va at taco.cc.ncsu.edu>, Chip Peele 
> <chip_peele at ncsu.edu>>wrote:
> 
> >I'm not so sure there is a secrete.  I typically do blunt and cohesive end
> >ligations at 37C for about 1 hour using my own buffers then transform.  I
> >nearly always get my clone.
> 
> >Gary
> 
> 
> Gary,
> 
> What kind of buffers are you using?  Just curious....
> 
> Victor



Victor,

The following protocol works equally well for blunt and cohesive
end ligations:

I begin by determining the number of picomole ends for vector and
insert using the following equation:

    (2x10e6)/(#bp x 660) = #pmol ends/ug DNA

Then I start with 200ng vetor in my ligation reactions (for both
blunt and cohesive end ligations).  I calculate how many pmol
ends is equivalent to 200ng vector DNA then use 1:3 ratio of pmol ends
(vector:insert) for cohesive ends and 1:5 ratio of pmol ends for
blunt ligations to determine nanograms of insert to use.  My
vector and insert DNAs are resuspended in sterile water.  The
DNAs are combined in an eppie and dried in a speed vac.  The
pellet is then disolved in the following:

    7.5 ul water
    1 ul 10x buffer
    1 ul ATP (10mM stock)
    0.5 ul T4 DNA Ligase (I use BM's)

Reactions (Blunt and Cohesives) are incubated 1-3 hrs at 37C then
transformed.

10X ligase buffer:
    600mM Tris pH 7.5
    50mM MgCl2
    10mM DTT
    15% v/v glycerol

Good luck,
Gary