Ligase free annealing question

redeamer 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
> news:p05200f12bc374c28626f@[192.168.80.1]...
> > >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
>  the
> > >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.
> 
> Cheers,
> Emir

Hello!

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
with DK.

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,

Drew



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