Improved method for the production of M13 phage and single-stranded DNA for DNA sequencing.
An improved method is described for the efficient production of M13 phage and M13 single-stranded (ss)DNA in a relatively short time period. Infection of E. coli (F') cells with as few as 5 phage particles can yield 10(12) phage particles/mL in 3 hours if the cells are grown in LB broth or SOB broth supplemented with about 5 mM Mg2+. The method tolerates large variations in the initial multiplicity of infection (5-5000 phage per 5 x 10(7) cells) and still yields about 10(12) phage particles/mL. These amounts are sufficient to purify 10-15 micrograms of ssDNA and to carry out at least 10-15 DNA sequencing reactions.
Reddy
P
P
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. prasad@enh.nist.gov
McKenney
K
K
D017104:Bacteriophage M13 / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D003470:Culture Media / Q000494:pharmacology; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000302:isolation & purification; D004279:DNA, Viral / Q000302:isolation & purification; D004926:Escherichia coli / Q000821:virology; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*
D003470:Culture Media; D004277:DNA, Single-Stranded; D004279:DNA, Viral