Frank O. Fackelmayer
Frank.Fackelmayer at uni-konstanz.de
Tue Feb 8 08:54:53 EST 2000
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.
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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