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| SINGLE STEP PROKARYOTIC RNA ISOLATION
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| [ 文章来源： | 文章作者：
| 发布时间：2007-01-09|
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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.
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