Recovery of episomal DNA from COS cells

Dave Smith DSMITH at UTU.FI
Wed Jul 20 05:24:07 EST 1994


As part of an expression cloning procedure we are trying to recover
episomal plasmid DNA from single COS-7 cells using the Hirt method. After
transfecting (by electroporation) the COS cells with the library DNA, the
cells are plated on glass chamber slides, cultured 36 hours and then fixed
with acetone and stained with an antibody against the molecule we are
trying to clone. The slide is then screened microscopically and positively
stained cells identified. The slide is then air dried and 1 ul of water
placed over the positive cell which is resuspended/broken into the water
using a micropipette tip. The cell suspension is then taken and put into
Hirt buffer and DNA extracted as in the normal Hirt method. The plasmid DNA
is used to transform E.coli by electroporation and will be used to
retransform COS cells.

In a mock experiment using a known cDNA diluted 1:10,000 into library cDNA
we can readily detect stained cells with an mAb against the protein encoded
by the known cDNA (control stainings are clean) but have so far been unable
to recover plasmid which on retransfection gives stained cells (we get some
plasmids back though). Has anybody tried this technique ?  We heard about
it 3rd hand from a source who didnt know where it came from, anybody knows
where it came from ? Any ideas why we are not be able to recover plasmids
that will retransfect and stain?

Dave Smith, PhD



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