Cohesive end ligation of dsDNA to ssDNA

Peter Ianakiev ianakiev at genome.wi.mit.edu
Thu Feb 14 12:24:56 EST 2002


Hi Emir
Before making the library bear in mind that ssDNA will be degraded in the
E.Coli, so you would have to fill the gap with T4 Pol. Otherwise I see no reason
why it should not work (if you have phosphorilated ends).
Good luck

Emir Khatipov wrote:

> Hi Dima,
> By 1) and 2) I meant to ligate those 1) and 2) together. So, I understand
> that T4 ligase
> would not ligate 2 ss oligos, but would catalyze 3-strand ligation, right? I
> sort of
> figured that ligases do not depend on 2 reaction sites as restrictases (why
> should they,
> after all?...). Did you try such a cohesive 3-strand ligation yourself?
> Should I check in
> a small scale if mine would work first, before ligating tons of DNA for the
> library? At
> last, is this _definitely_ T4 ligase that would catalyze such a reaction?
> Any references?
> Emir
>
> "D.K." <dk at no.email.thankstospam.net> wrote in message
> news:9u6tgg$dgk$1 at news.doit.wisc.edu...
> > In article <X8CN7.89$t4.2591 at news.uchicago.edu>, "Emir Khatipov"
> <khatipovNO at NOuchicago.edu> wrote:
> > >I think I have a nontrivial question here for cloning gurus.
> > >Would ligase (T4 or Ec, or RNA ligase) ligate the following molecules to
> > >each other:
> > >1) vector linearized with 2 different 6-cutter restrictases (4-base
> > >overhangs),
> >
> > Basically, no.
> >
> > >2) single-strand (ss) oligo with ends complimentary to the sticky ends on
> > >the linearized vector?
> > >vectorxxx    <--tgca-random-oligo(50bp)-ttgg--> xxxvector
> > >vectorxxxacgt
> aaccxxxvector
> >
> > Yes.
> >
> > >So the question is does the 3-strand ligation work, and if work, how
> > >efficiently?
> >
> > IMO, about the same as regular sticky ends.
> >
> > DK




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