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