We've used a method by Shaw and Clewell, (1985) J. Bacteriol.
164:782-796. It uses cold sodium azide for spinning down the cells so is
pretty nasty. There is another method by Mayford and Weisblum (EMBO I
think) but I don't have the reference handy. It spins the cells in Tris,
EDTA and 100 mM BME.
Getting rid of rRNA is more difficult. You could try spinning the ribs
out by ultracentrifugation (Make and S-150), but this usually involves
French Pressing and high salt to keep ribs intact, so don't know what it
might do to your mRNA.
On Sat, 13 Mar 1999, Emir Khatipov wrote:
> I would appreciate any guides on the following:
>> 1) How to rapidly quench BACTERIAL cells for subsequent RNA isolation?
> Convential cell harvesting by centrifugation is too long. Would treatment
> with trichloracetic or chloric acid work? Alkaline treatment?
>> 2) How to separate BACTERIAL mRNA from total RNA (mRNA+rRNA)? I.e. how to
> get rig of rRNA?
> Bacteria DO NOT HAVE poly-A tails, thus commercial kits based on
> poly-A-matrix binding do not work. In fact, there are poly-A in bacteria,
> but not all mRNA have them.
>> I need mRNA for expression analyses.
>> Thank you.
Elizabeth Rogers Department of Biological Sciences
Phone:(410) 455-2285 University of Maryland Baltimore County