From jwbrown@crab   Wed Jan 15 02:45:08 1992
Subject: SINGLE STEP PROKARYOTIC RNA ISOLATION 

       In the past, poly(A)+ RNA has not been detected in prokaryotes.  
In the early 80s, a new method of isolating RNA from bacteria was 
developed, involving lysis by protease K in the presence of SDS and 
potent RNase inhibitors (heparin & 1,10 phenanthroline) and without 
phenol:chloroform extractions.  This method has allowed the detection of 
poly(A)+ RNA in a variety of prokaryotes: Bacillus subtilus, B. brevis, 
B. polymyxa, E. coli, Rhodospirillum rubrum, Rhodopseudomonas capsulata, 
Caulobacter cresentus, Hypomicrobium B-522, Anabena variabilis and 
Nostoc MAC.  It is possible that the phenol:chloroform extractions used 
in earlier procedures selectively eliminates poly(A)+ RNA or mRNA, 
although this is not the case when isolating RNA from eukaryotes.  RNAs 
prepared by this method are very crude, but can be used directly for 
oligo(dT)-cellulose chromatography or urea-agarose gel electrophoresis, 
or can be further purified by any method NOT including organic 
extraction.
        RNase-free technique should be used throughout this procedure, 
in spite of the RNase inhibitors.



Lysis buffer (per 200ml)      final conc.     working conc.
   1.9380 grams Tris, pH 7.5      80 mM          35.6 mM
   0.4067 grams MgCl2-7H2O        10 mM           4.4 mM
  140 ul 2-mercaptoethanol        10 mM           4.4 mM

  5% SDS - 1 gram per 20ml

  2.3 mg/ml Protease K - 46 mg in 20ml ddH2O

  0.1M EDTA - 4ml 0.5M EDTA + 16ml ddH2O

  20mM 1,10-phenanthroline - 79 mg in 20ml ddH2O
      (boil to dissolve)

  2 mg/ml Heparin - 40 mg in 20ml ddH2O

  2.5M Na acetate - 68 grams per 200ml

Filter sterilize protease K, 1,10-phenanthroline and heparin.



1. Pellet 1.5ml stationary phase or 3ml log phase cells in a microfuge 
tube.  Decant the supernatant and remove residual droplets from the tube 
with a pasteur pipette.

2. Resuspend the pellet in 200ul lysis buffer.  Add 45ul each of the 
following in sequence:

        2 mg/ml heparin
        20mM 1,10-phenanthroline
        0.1M EDTA
        5% SDS
        2.3 mg/ml protease K

3. Vortex and incubate 20 min. at 37C.  (Some species may require 
sonication prior to proteolysis to lyse the cells, i.e. Bacillus)

4. Add 50ul 2.5M Na acetate and 1ml EtOH.  Vortex, and incubate ON -20C.

5. Pellet 15 min. and resuspend in 20ul AN.  Store at -20C.


NOTE: For oligo(dT)-cellulose chromatography, omit steps 4 & 5, but add 
50ul 5M NaCl and chromatograph immediately, or freeze on dry ice and 
store at -70 for future chromatography.



Gopalakrishna, et al 1981 NAR \9(14)\:3545

JWBrown (unpublished data)
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