Trichloroacetate and nucleic acids

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Tue Mar 23 15:51:50 EST 1999


In article <F910vo.215 at midway.uchicago.edu>, "Emir Khatipov" <ekhatipov at NOuchicago.edu> wrote:
:Does anybody know whether trichloroacetate will destroy DNA and (more
:importantly) RNA if used for rapid quenching of bacterial cells for further
:analysis.
:
:The story is that I need a way to very rapidly and quantitatively isolate
:total bacterial RNA. Conventional methods requiring centrifuging off cells
:are too slow - seconds matter, and 1 min. will be too long. Since
:biochemists use trichloroacetate of chlorate to denature protein and thus
:instantly kill the cells, I thought that those acids can be useful. However,
:I am not shore that the acids will not destroy (hydrolyse,...) NAs. What
:about alkali?
:
:I will appreciate everybody's considerations on the matter. Quenching with
:azyde does not look a solution, azyde will inhibit respiration, but not
:nucleases...

Hi, Emir! :-)

TCA is definitely not suitable. Here is an old and working alternative I 
can think off: 

Freeze in LN2 (better yet, in ethanol/CO2), grind with a pertel and 
mortar while frozed in LN2 with broken glass or sand - this will 
definitely work for braking E.coli (works even for sturdy fungi and 
algae), when LN2 _just_ evaporates add whatever quenching solution
to kill off nucleases (I'd go with SDS/EDTA + RNAase inhibitors, but
your olde phenol/chloroform should work as well). 

Voila! You have stopped metabolysm intantaneously, and have cell
extract with all hydrolytic enzymes killed off... 

How about that? 

        - Dima

P.S. A while ago, I got an email from you but my reply kept returning (?)


 



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