R17 RNA replicase. VI. A large scale preparation of R17 template RNA.
A new procedure for R17 RNA preparation was devised using a 300 liters fermenter. The key factor in processing such a large quantity is the purification of phage particles prior to RNA extraction. The method involves the preparation of 250 liters of crude lysate, condensation of phage particles by the partition method, purification by DEAE-cellulose column, and removal of adherent proteins by a series of high-salt washes. The method permits preparation of approximately 3 g of phage particles free of ribosomal fragments and RNase. Phage RNA extracted in gram quantities using conventional methods often contain phenol. Thus repeated extraction of R17 RNA with salt-alcohol mixture is required.
D003090:Coliphages / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008722:Methods; D010636:Phenols; D012367:RNA, Viral / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D013698:Templates, Genetic; D014461:Ultracentrifugation; D014779:Virus Replication
D010636:Phenols; D012367:RNA, Viral; D012967:Sodium Dodecyl Sulfate; D012321:DNA-Directed RNA Polymerases