RNA prep from gram+ bacteria???

harald at mol.univie.ac.at harald at mol.univie.ac.at
Thu Mar 26 13:48:17 EST 1998


Hi, netters!

I want to prep RNA from gram+ bacteria. To do so, I tried a modification of the Chom & Sach method which is 
quite similar to the TRIAZOL (other brand names TRISOLV, RNASOLV) procedure. While gram- cells lysed rapidly (I 
used E. coli O26B6, a wild-type strain) and gave very pure RNA (260/280 about 2.1) with satisfying yields 
(about 1000 µg from 10 ml o/n culture brew), this procedure failed completely with gram+ bacteria. As a 
consequence, I tried sonication (3x 20 sec, about 30 W) of the bacteria in the TRISOLV reagent. However, 
sonication was ineffective. Other approaches included cell wall digestion with lysozyme and prep using Quiagen 
RNEasy spin columns as well as TRISOLV, both preps failed. Our latest trial included digestion with lysozyme 
and mutanolysine followed by proteinase K digestion. However, also this one failed.

Are there any people who prep total RNA from gram+ bacteria (e. g. Bacillus subtilis) on a more or less regular 
basis, or who have at least some experience with this topic? ANY bit of info is welcome!

Harald F. Sigmund




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