oligo cloning

Csaba Kiss csakis at ki.se
Tue Feb 8 04:04:43 EST 2000


Hi!
I agree with all the others that the CIP treatment should be done carefully.
Calculate your "DNA ends in picomol". You can find the easy conversion in
Stratagene's catalouge, but I am sure you can find it at many places.
The othe important point is that SmaI is a notoriously bad enzyme. Try to
use as little as possible. Digest your sample at room temperature, only for
30 minutes, then if I were you i would phenol->chloroform->Et-OH it. The
SmaI can create overhangs instead of blunt ends.

I hope it helps.

Csaba

"Caroline Szymeczek-Seay" <css at med.unc.edu> wrote in message
news:389F1C35.2B4148CC at med.unc.edu...
> 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






More information about the Methods mailing list