Extraction of RNA

A 100-ml sample of fresh cell culture was rapidly poured onto 100 ml of crushed，frozen extraction buffer containing 80 mmol/L Tris-chloride（pH 7.5），10 mmol/L MgCl2，10 mmol/L β-mercaptoethanol（freshly added），25 mmol/L NaN3，and chloramphenicol（200 μg/ml），rapidly mixed，and centrifuged at 7，000 rpm for 3 min.The cells were lysed by adding 5 ml of lysis buffer containing 0.5%sodium dodecyl sulfate，10 mmol/L EDTA，2 mmol/L O-phenanthroline，0.2 mg of heparin per ml，and 0.25 mg of proteinase K per ml and then frozen in a dry ice-ethanol bath and thawed at 37℃for three successive cycles.The cell lysates were incubated at 37℃for 20 min and then extracted with hot（65℃）phenol（redistilled）-chloroform（1∶1，vol/vol，three or four times until a clear aqueous phase was obtained.DNA and RNA were precipitated by adding 2 volumes of ethanol to the aqueous phase and dissolved in 5 ml of 50 mmol/L Tris-chloride-5 mmol/L MgCl2（pH 7.8）.DNA was digested by incubation with RNase-free DNase I（50 μg/ml）at 37℃for 20 min.DNase I was removed by phenol extraction，and the RNA was finally collected by ethanol precipitation.About 1 mg of RNA was obtained from 100 ml of cells.The ratio of absorbancy at 260 nm to that at 280 nm was 1.8 to 2.0.RNA was used either for dot blots（5 μg per dot）after denaturing with formaldehyde or for Northern blot（15 μg per lane）hybridization after glyoxylation and agarose gel electrophoresis.(https://www.daowen.com)