Cohesive end ligation of dsDNA to ssDNA
ianakiev at genome.wi.mit.edu
Thu Feb 14 12:24:56 EST 2002
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).
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?
> "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
> > 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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