Ligase free annealing question
alion85 at hotmail.com
Sat Jan 24 06:15:45 EST 2004
"EK" <nobody at elnino.com> wrote in message news:<sPnQb.50$_4.22209 at news.uchicago.edu>...
> ""Michael L. Sullivan"" <mlsulliv at facstaff.wisc.edu> wrote in message
> > >Thanks Hiranya,
> > >For annealing with the vector, I will not use ligase and rely solely on
> > >annealing of complementary sticky ends on the vector and the inserts. The
> > >reason for it is that I believe it will reduce the anount of self ligated
> > >vector. I do get clones of self ligated vector if I use ligase, despite
> > >noncompatible ends.
> > >Emir
> > What I've generally done for cloning oligos like this is to NOT
> > kinase the oligos, then use a few levels of annealed oligo insert in
> > the ligation reaction, like 3X, 10X, and 30X molar excess over
> > vector. Since the oligos aren't phosphorylated, you don't recover
> > clones with multiple inserts, but you can still favor getting clones
> > with insert by having a lot of it in the ligation reaction.
> > As for self ligated vector despite non-compatible ends, it has been
> > argued in this newsgroup before that this can be due to incomplete
> > digestion with both enzymes, with religation of single cut vector a
> > favorable event. If there is another enzyme that cuts between the
> > two you are using for your cloning, you could actually cut vector
> > with three enzymes to reduce this possibility.
> > What vector are you using for this cloning? Is there color
> > selection. This would allow you to ignore religation events due to
> > single cuts.
> It is an unusual cloning strategy. The plasmid vector is pre-cut with the
> blunt-cutting restrictase and then the sticky ends are generated by T4
> polymerase in the presence of only one nucleotide. The polymerase removes
> one strand all the way until it gets to the first letter matching the
> nucleotide in the reaction. In my case, 15nt sticky ends are produced on
> both ends of the linearized vector. Inserts encode short peptides, and once
> inserted into the vector, the peptides can be expressed in fusion with an
> enzyme. It is basically an ELISA-based screening system for peptide ligands.
> No color selection.
> Anyway, I think you guys answered my initial question and I appreciate your
> advises. I just should try several dilutions and ratios of insert and vector
> to find optimal conditions for one or two samples and use those throughout.
> It would not be feasible though to do dilutions for every case, because the
> idea is to clone and screen hundreds of peptide sequences at a time at a
> time, possibly using lab automatics.
Emir, why dont you dephosphorylate your vector like Hiranya suggested,
and just ligate the oligos into you vector? Anyway ligase wont
interfere with any of your downstream processes i think, fully agree
I have some suggestions about T4-removing single strand DNA, make sure
you are doing it at low temperature, i dont remember how low it should
be, some 14 or 12 degrees, check it out in a tech.. cause this enzyme
is such a reactive pacman that could eat everything you need to remain
in you vector:-))))
my recipe for doing oligos is ideal for gel shift assays, and i dont
see any reason why it shouldt work with cloning...
Have a nice cloning,
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