oligo cloning

Colin Rasmussen 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.

Colin





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