Help with Lysis Buffer 4 adherent cells please !

[Mart Speek]

runting at wehi.edu.au wrote:
: HI there

: I am in need of some suggestions for a new recipe for
: lysis buffer to lyse adherent mammalian cells from
: 96 well tissue culture plates.

: The lysis buffer I am using at the moment is 30mM Tris,
: 100mM EDTA with 20% Sarkosyl. I am getting unreliable
: results using this at the moment.

: Any suggestions as to a new lysis buffer which might
: work better in triated thymidine uptake assays which
: I am using would be greatly appreciated

: Thanks in advance,

: Andrew Runting
: Ludwig Institute for Cancer Research
: Melbourne Australia
: runting at licre.ludwig.edu.au
----------------------------------------
Dear Andrew,

Although you don't say what is your problem, I would recommend to
use the following lysis buffer:

50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl2, 0.5%(v/v) Nonidet P-40
and 1 mM DTT. Add protease, RNase inhibitor(s) if necessary. Note that 
the concentration of Nonidet can be increased/decreased depending
on your needs. This coctail guarantees 100% lysis for most monolayers.
I have used it for RNA/protein fractionations.

Hope this helps,

Mart

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