rescue plasmids from single COS-7 cells
daniel.snell at bbsrc.ac.uk
Thu Oct 7 11:22:39 EST 1999
Matthias Liniger wrote:
> I transfected COS-7 cells with a plasmid library.
> I tell you: It's not trivial to scrape away single positive
> (immunoscreened) cells, isolate plasmids
> and electroporate into E. coli.
> Any Ideas?
Hi there, we've used COS-7 cells many times for cDNA screening and in
all that time I don't think I've ever managed to scrape just the one
positive cell. As long as you're not too clumsy and obliterate half the
slide it doesn't really matter, the cDNA wanted will still be greatly
enriched. The method we use is to grow the cells on microscope
slides, do your standard immunohistochemistry and then scan the slide
wet, covered in a cover slip as normal, recording all the positions of
stained cells using the scopes vernier scale. Then remove the cover
slip and allow the slide to dry, go back to your positives and using a
gel loading tip cut back so the end is approximately one cell thick
(bit of guesswork and a sharp acalpel blade) drop 2-3 ul of water onto
the cell, scrape the cell (and as few surrounding cells as possible into
this drop, and aspirate the drop and put straight into the Hirt
extraction solution (10mM EDTA, 0.6% SDS, 100ug/ml proteinase K). This
works for us with a success rate of about 50%, just needs a lot of
Alternatively of course, if you're certain the protein you're staining
is expressed on the COS cell surface then you could remove the cells
usin EDTA and use one of the magnetic sorting techniques to remove the
positive COS cells. You'll never just get one cell, but again for cDNA
screening it doesn't really matter.
hope some of this helps
Institute for Animal Health
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