Rapid small-scale isolation of SV40 virions and SV40 DNA.
A rapid method for the small-scale isolation of SV40 virions and SV40 DNA is presented. CV-1 monkey epithelial cells are transfected with linear SV40 DNA. After the onset of transfection, cells are lysed by several freeze/thaw cycles and virions are isolated using polyethylene glycol (PEG) precipitation of DNase I treated lysates. Viral DNA is released by proteinase K and dithiothreitol treatment of the isolated virions followed by phenol/chloroform extraction and ethanol precipitation. This method yields on average 7.5x10(4) plaque forming units (PFUs) and DNA of adequate purity and concentration to be used for restriction analysis on ethidium bromide agarose gels from a single 35-mm tissue culture dish.
Orlando
S J
SJ
Department of Biological Sciences, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, CA 90840, USA.
Nabavi
M
M
Gharakhanian
E
E
D000818:Animals; D002460:Cell Line; D002725:Chloroform; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D004229:Dithiothreitol; D019286:Endopeptidase K; D000882:Haplorhini; D009154:Mutation; D019800:Phenol; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection; D014771:Virion / Q000235:genetics / Q000302:isolation & purification*; D014773:Virology / Q000379:methods*
D004279:DNA, Viral; D019800:Phenol; D002725:Chloroform; D019286:Endopeptidase K; D004229:Dithiothreitol