watchcity at my-deja.com
Tue Feb 8 08:43:02 EST 2000
Hello Caroline, What results are you getting from your digested, and
un-phosphatased, vector only ligation control? Try rekinasing the CIPed
vector to see if you regain this level of ligation. Do you have PCRable
oligos for the vector that span the insertion site? You can use these to
test for insertion, orientation, and # of inserts. (BTW, if you have multiple
inserts and the 2 end sequences are in the correct orientation, a single
unique restriction site will clean out the extras (with that digest, and
something like HighPure silicon matrix for dna purification; some products
won't bind under 100mers.). When you have a linker made with oligos that have
incompatable sticky ends, multimers always can be cleaned up in this
fashion.) There is a shrimp alkaline phosphatase that is highly
thermo-labile, and works well. If you use dna purification kits to change to
the proper buffer at each step, the results may be better. Good hunting,
In article <389F1C35.2B4148CC at med.unc.edu>,
Caroline Szymeczek-Seay <css at med.unc.edu> 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.
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