Biochemical quantitation of PM2 phage DNA as a substrate for endonuclease assay.
Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4 degrees C; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.
Joo
Yoo Jin
YJ
Laboratory of Biochemistry, School of Life Sciences & Biotechnology, and BioInstitute, Korea University, Seoul 136-701, Korea.
Kim
Hee-Ju
HJ
Lee
Jae Yung
JY
Kim
Joon
J
D019240:Corticoviridae / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology; D004270:DNA, Circular / Q000302:isolation & purification / Q000378:metabolism; D004279:DNA, Viral / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D016679:Genome, Viral; D005990:Glycerol / Q000494:pharmacology; D012034:Refrigeration; D014316:Tritium / Q000378:metabolism; D010948:Viral Plaque Assay
D003451:Cryoprotective Agents; D004270:DNA, Circular; D004279:DNA, Viral; D014316:Tritium; D004720:Endonucleases; D005990:Glycerol