css at med.unc.edu
Thu Feb 10 12:27:37 EST 2000
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
> * 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,
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