Ligation problems

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Fri Jul 31 06:19:41 EST 1998


Ian A. York wrote:
> 
> We have purified some genomic DNA and we want to put it into an expression
> vector.  We have sheared it, blunted, ligated BstXI adaptors, and ligated
> into complementary BstXI sites (Invitrogen vector, Invitrogen adaptors).
> Result--very inefficient ligation. .....


two points come to my mind:
* is your DNA clean enough to be sure you don´t have residual phosphatase
activity? If your fragments become dephosphorylated, many of the effects you
describe would be explained.
* was your DNA really blunted? What enzyme did you use for blunting? I think
that when you shear DNA you get quite long single stranded ends that might be
difficult to make blunt.

To investigate if either of these points may be the problem, add ligase and
buffer to an aliquot of your DNA (without vector), let stand at room temp
overnight, then analyse on an agarose gel along with a sample without ligase.
If your DNA is good enough for cloning, you should see efficient ligation
(formation of a high molecular weight smear with average fragment size MUCH
bigger than in the sample without ligase).

If you don´t see ligation, repeat the experiment, but add T4 polynucleotide
kinase to the overnight ligase reaction. The kinase works very well in ligase
buffer, but add 1mM ATP in addition to what is in the ligase buffer. If you
get ligation then, the problem is dephosphorylation by residual phosphatase activity.

If you still don´t see ligation, try a different method to make DNA blunt. I
have used mung bean nuclease for that some years back, but there are certainly
better methods available today.

Continue with cloning only after you get efficient ligation of the genomic DNA fragments.

Hope this helps,
Frank



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