Jack's

Thomas Delohery t-delohery at ski.mskcc.org
Wed Apr 5 08:40:47 EST 1995


Dude, I'm a fan.  I also thought sort blots would "revolutionize the
        field".  I was able to get someone to try it once, only once.
        The guy had so much hemolysis in his sample the blots were
        visible from the hemoglobin.  Not much use and he never tried
        it again.
        We did use your cocktail of RNAase inhibitors to suspend fixed
        cells stained with Chromomycin to isolate G1,G2,early and late
        S-phrase fractions.  We had good Northerns from 1 million cells
        per fraction.  Sorting fixed myoblast cells was a nightmare -
        they were torn apart and trying to show sort purity results was
        very problematic.  We went to your paper after we tried the
        protocol Dennis Young cited (below).  The 5XSSC caused all the
        cells to clump into huge aggregates.  We did sort into the
        5XSSC but we couldn't suspend the cells in it.

        Cheer up - maybe it was an idea (assay) too far ahead of it's time.
        FURTHUR.

- From Dennis Young:
Paranoia of RNAse's will help ensure good results. DEPC-treated sheath fluid, as
well as vigilant sample-tubing cleaning are a must.
See Cytometry 11:869-874 (1990).
(They used fixed cells, 5XSSC instead of the standard PBS and washed
sample-tubing with 10% Hydrogen Peroxide.)


tom d.

  QUESTION AUTHORITY..............
        on every fundamental precept proposed as an explanation of any
       aspect of reality; because reality is a perception and therefore
       inherently subjective and gaussian in distribution. (possibly Poisson)

        (I said that.)
--
==============================================================================
 Thomas Delohery                        | Internet: t-delohery at ski.mskcc.org
 Manager, Flow Cytometry Core Facility  |    Phone: (212) 639-8729
 Memorial Sloan-Kettering Cancer Center |      Fax: (212) 794-4019
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