colin at pombe.usask.ca
Mon Feb 7 17:03:02 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.
What I would do is to use unkinased oligos, and leave the phosphate on the
vector. Then ligate with diminishing amounts of vector while keeping the
insert concentration the same. Do a plating onto X-gal plates and pick
white colonies. With your way, you run the risk of inserting multiple
copies. The way I describe, you can only get one insert per vector.
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