Preparative-scale isolation and purification of procaryotic and eucaryotic ribosomal 5 S RNA: Bacillus subtilis, Neurospora crassa, and wheat germ.
Ribosomal 5 S RNA from three different organisms has been isolated in high yield and purity. Without prior isolation of ribosomes, a presoak in buffer followed by phenol extraction, DE-32 ion-exchange chromatography, and Sephadex G-75 gel-permeation chromatography yields at least 5-10 mg of electrophoretically homogeneous 5 S RNA from 100 g of cells. Ribonuclease activity is eliminated by various combinations of low temperature, sodium dodecyl sulfate, phenol, and bentonite. High-molecular-weight contaminants are suppressed by either 65 degrees C heat treatment or lowered sodium dodecyl sulfate concentration. For the eucaryotes, 5.8 S RNA contamination is reduced either by low temperature in the initial solubilization or by postponing 65 degrees C heat treatment until after the phenol extraction step.
D001412:Bacillus subtilis / Q000235:genetics*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008722:Methods; D009491:Neurospora / Q000235:genetics*; D009492:Neurospora crassa / Q000235:genetics*; D012329:RNA, Bacterial / Q000302:isolation & purification; D012331:RNA, Fungal / Q000302:isolation & purification; D012335:RNA, Ribosomal / Q000302:isolation & purification*; D012343:RNA, Transfer / Q000302:isolation & purification; D014908:Triticum / Q000235:genetics*
D012329:RNA, Bacterial; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D012343:RNA, Transfer