TCA precipitation and cell harvesting
WHITSITT, MARK STEVEN
msw1633 at summa.tamu.edu
Tue Aug 20 19:39:31 EST 1991
In article <5300 at beguine.UUCP>, danielg at med.unc.edu (Daniel Gene Sinclair) writes...
>Couldn't we just add the tca to the cells first? Wouldn't *that* cause
>them to detatch? Why scrape them in PBS? Does free DNA like to stick to
>the plastic plates or something? I don't like the scraping, it is
>awkward, time consuming, and sloppy. Anyone know why everyone does it
The point of TCA precipitation is to get the nucleic acid to "bind" to the
filters, allowing you to wash away any unincorporated radionucleotides (ie. only
the large nucleic acid molecules are "acid precipitable") and then count the
number of counts incorporated into the DNA/RNA. If you add TCA to the
cells, you will precipitate the nucleic acid into the dish and never be able
to separate large molecules from the unincorporated stuff, and your experiment
will mean nothing.
Molecular biology at the bench is work intensive, boring and time consuming.
Guess you're finding out what the rest of us did when we started.
Oh, and another thing! Odds are that the protocol you received from wherever is
probably pretty close to optimum. There might be changes that would improve it
somewhat, but is the improvement great enough to justify the time spent trying
to re-optimize. Another general rule in Mol Bio is,at least to my thinking is
unless you are 100% sure that you can make a protocol better, easier, faster,
etc., without reducing the quality of the results, you are probably better off
not messing with it and just getting the work done. --- this, btw, is my own
sentiment, not necessarily that of my maj prof.---
good luck and DONT add TCA to the cells
Mark S. Whitsitt, N5RJF Texas A&M University, Dept of Biochemistry
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