3H Thym incoproration assay?

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Tue May 30 11:02:45 EST 2000


In article <3933DF51.86D507AD at giccsX.georgetown.edu>, Will <
WillX at giccsX.georgetown.edu> wrote:
>Hello all,
>    I know this is real base question, but here goes anyway.
Does
>anybody have a nice easy protocol for 3H thymidine
incorporation into
>monolayer cells. I've looked around and can't find the info I
need.
>Most of it is straight forward, my only questions are; a)
should I lyse
>my cells before TCA precipitation (ie can I wash excess 3H
Thymidine and
>precipitate on the plate before lysis? b) if not, is a standard
SDS
>lysis buffer O.K. for running the precipitation? Thanx in
advance for
>any help.
>
>

The TCA will do a simultaneous lysis/pptn.

We do the following for adherant cells in 24 well plates:

Wash 2-3x w/ PBS

Add 1 ml ice cold 10%TCA to each well. Sit on ice for at least 20
min.

Wash 2-3X 10%TCA

Wash 2x ethanol/ether (2:1) - IN THE FUME HOOD!

Air dry (e.g. 20-30 min. in the hood)

Add 0.5 ml 1 N NaOH per well to solubilize (This can be
conveniently done by wrapping the plate in parafilm and put on a
shaker overnight.)

Count 100-200 ul.


Nick Theodorakis

nicholas_theodorakis at urmc.rochester.edu
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