ss-DNA-Ligation

Zophonias Oddur Jonsson zjons at rhi.hi.is
Fri Mar 4 12:12:24 EST 1994


In <MAILQUEUE-101.940304124238.384 at bch.univie.ac.at> DORIAN at bch.univie.ac.at (Dorian Winter) writes:

>Hi, Netters,

>We d like to ligate ss-DNA oligos to the 5 -overhang of our vector.
>This happened, we need to fill up the single-strand, creating a blunt
>end.
>Our questions are:
>1. Is this possible?
>2. Which is the most suitable ligase and buffer-system?
>3. Which is the polymerase of choice?

>Any suggestions are welcome!

I doubt that any DNA ligase would recognize your template as ds-DNA unless
you have a very long overhang but you can't be sure without trying.

Good old T4 ligase in standard reaction buffer, with a bit of PEG or another
polymer is probably the best.  And then of course there is the bunting.  It
should work OK with good old Klenow.

But why don't you just create a linker by making (or buying) the complementary
oligo as well?  Then I'm sure the ligase will do it's job and you don't have to
worry about the blunting.  

Just an idea!
Zophonias

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Zophonias O. Jonsson		    SOME people's talents are so well hidden
University of Iceland                  that you overlook them completely!
zjons at rhi.hi.is					     -Stephen Young
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