4 fragment ligations.....?
clarosa at biocomp.unl.edu
Wed Aug 23 09:56:21 EST 2000
> I use 5 fmol of vector and 10 fmol of each insert fragment (including
> double-stranded oligos--some people seem to use way too much) in a 10 ul
> ligase reaction. I estimate concentrations by running a bit of each
> fragment on an agarose gel and comparing the staining to a standard.
> Optimal concentrations for 2-fragment ligations are discussed in Table
> 3, "A practical set of recommendations for ligation", in Revie, et al.,
> Nucleic Acids Res 16:10301-10321, 1988, "Kinetic analysis for
> optimization of DNA ligation reactions". Worth reading the whole paper
> As to the "chain reaction cloning", I don't know.
Thanks for the responses. I have looked at this chain reaction cloning
procedure. It uses thermo stable ligases and oligonucleotides where
the oligonucleotides bridge the pieces to be ligated. It appears very
interesting but the method is just too new for me to trust until I see
some examples other labs using it. It looks like it might be something
that some company would develop into a kit if it really is reliable
(Ampligase by Epicentre was mentioned in the paper.. I did not see any
application notes in the Epicentre web site concerning use of ampligase
in chain reaction cloning.)
I will probably try to ligate the 3 insert fragments first, size select,
check the fragment by pcr, then ligate into the vector....I spent
several hours doing library and web searching for papers discussing 3
and 4 fragment ligations.... there are a few patents where it is
mentioned for 3 but I could not find a protocol detailing the fine
points of 3 or 4 step ligation. If anyone runs across that perhaps you
can post it.
Regards.... PC LaRosa
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