Help!!!! how to clone a PCR fragment?
hmmoss at MAIL.MED.CORNELL.EDU
Thu Aug 3 17:29:13 EST 1995
I have a cloning project that involves inserting a PCR fragment with one
cohesive (Bam HI) and one blunt end into pBS. I can foresee two problems
with this, namely: 1) the presence of the nontemplate specific A residue
that is added at the end of PCR fragments, which gives me a non-blunt
end, and 2) the absence of the 5' phosphate of the primer on the "blunt"
end. I know the theory about what should be done to eliminate these
problems, but what I need is someone with a little experience doing
this. I'll start with the nontemplated A issue: I've heard that
incubation with DNA pol I will "polish" the end by the activity of the
exonuclease. Does anyone have practical experience with this, and is
this even the best way to go about this? If so, can one accomplish this
in the PCR buffer including dNTPs?
Second, I've imagined that the T4 PNK reaction would ocuur more
efficiently if I phenol extract the DNA before the PNK reaction. True?
Finally, if phenol extractions are necessary, I'm going to be
concerned about loss of sample, so the method that accomplishes the goals
with the fewest extractions is the most valuable to me.
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