s.hopkirk at virgin.net
Fri Feb 11 14:19:59 EST 2000
Hi, just wondering if you would mind forwarding on to me this protocol.
Caroline Szymeczek-Seay wrote:
> thanks for your answer! I'm trying a similiar protocol to yours that
> was emailed to me privately. Your advice is good, and very practical.
> "Frank O. Fackelmayer" wrote:
> > Caroline Szymeczek-Seay wrote:
> > > I'm cloning (or trying to clone) a double stranded blunt ended oligo (a
> > > 47mer) into pGL3 enhancer vector from Promega. Vector is cut with SmaI
> > > and CIPed. Oligos are kinased.
> > >
> > > My question: is there a rule of thumb for amount of oligos to add to
> > > vector? I'm trying a 10-fold molar excess, a 5-fold molar excess and an
> > > equimolar amt of oligo relative to vector. I'm getting no positives.
> > > This is a little hairy also because there's no blue/white screening for
> > > this vector.
> > >
> > > caroline
> > Hi Caroline,
> > I´ve done oligo ligations quite often, and for my lab this procedure works
> > well:
> > * dissolve UNKINASED oligos to 100pmol/ul in TE
> > * mix 5ul of both oligos, heat to 95C for 5 min, then cool slowly
> > * in a separate tube, precipitate 1ug of your cut vector with EtOH, allow
> > pellet to air-dry (do NOT dephosphorylate!)
> > * dissolve pellet in annealed oligos
> > * add 1.2ul of ligase buffer and 1ul of ligase (1U), then let stand
> > overnight at RT
> > * in your particular case, I´d add 10units of SmaI into the ligation mix to
> > cut vectors that have religated without taking up an insert. (of course
> > this works only if your oligo ends to not reconstitute a Sma-site, i.e. are
> > not 5´ GGG.....). During the overnight reaction, you´ll get more and more
> > vectors that have taken up an insert.
> > If you calculate it, you´ll see that the excess of oligos over vector is in
> > the 500-1000 fold. This is ok. We routinely get more than 90% clones with
> > exactly one insert. Remember that, as always, cloning success also depends
> > on the quality of your competent cells. Don´t use cells with competence
> > less than 5x10e8 (electroporation is usually good) for all cloning work
> > except for the most simple sticky-end subclonings.
> > Hope this helps,
> > Frank
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