hubahopp at gmx.de
Thu Feb 19 10:49:11 EST 2004
in my experience and opinion the crucial step is the purification of the
pcr product: if you EtOH precipitate, there will always some Taq and dNTPs
that coprecipitate, that then could do the damage. Or even worse, some
contaminants like DNAse (you write about loosing the Sfi site) could be
carried over once they are in the system.
As long as you can make sure there is no DNAse anywhere (incubate
linearized plasmid with aliquots of the solutions to be tested overnight at
37degC and then look for smear on a gel), you might consider gel filtration
columns (like eg the pcr purification kit from quiagen). Since they are a
time and resource consuming step in HTS, you might check the suitability in
a small scale experiment on the bench first.
When you prepare the vector, it could make sense to include a
phenol/chloroform purification step there before the gel filtration. Self
ligation background (in reality I think carry over of uncut vector
contrbutes a lot) can be minimized by transferring the digest to a new cup
without disturbing the mix. The uncut bugs sit on the wall and in the
niches along the lid and you'll set them free when you snip/vortex and
centrifuge the tube.
If you are not sure about Sfi I, Ngo MIV (sometimes named Ngo MI, eg from
NEB) could be an alternative. In my hands it worked better, maybe because
it cuts well at 37 degC in contrary to Sfi I.
Best of luck,
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