Two-step isolation of bacterial ribonucleic acid without using a chaotropic agent or cesium chloride centrifugation.
A simple RNA isolation method was developed to purify bacterial RNAs from a large number of samples simultaneously in an hour. The method is based on boiling the cells in the presence of Triton X-100 and lysozyme, and then preferential RNA precipitation with ammonium acetate. There is no CsCl centrifugation required. For the nitrogen-fixing bacterium Klebsiella pneumoniae, the depression condition can be maintained during the cell-harvesting process. The intact isolated RNAs appeared to be free of protein, with a yield of 100 micrograms RNA from a 4-ml cell culture of 100 Klett units (10(9) cells/ml). Any DNA present was in a form that did not react with a nifH probe following Northern blotting to nitrocellulose (i.e., was not single-stranded).
Chang
C L
CL
Department of Biochemistry, Kansas State University, Manhattan 66506.
Shi
B Q
BQ
Davis
L C
LC
D000085:Acetates; D015152:Blotting, Northern; D002499:Centrifugation, Density Gradient; D002586:Cesium; D011232:Chemical Precipitation; D002712:Chlorides; D003902:Detergents; D004587:Electrophoresis, Agar Gel; D007711:Klebsiella pneumoniae / Q000235:genetics; D008722:Methods; D009113:Muramidase; D017830:Octoxynol; D011092:Polyethylene Glycols; D012329:RNA, Bacterial / Q000302:isolation & purification*
D000085:Acetates; D002712:Chlorides; D003902:Detergents; D012329:RNA, Bacterial; D002586:Cesium; D011092:Polyethylene Glycols; D017830:Octoxynol; D009113:Muramidase; C028019:cesium chloride; C018824:ammonium acetate