A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column.
A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.
Reddy
K J
KJ
Division of Biological Sciences, University of Missouri, Columbia 65211.
Kuwabara
T
T
Sherman
L A
LA
D010582:Bacteriophage lambda / Q000032:analysis*; D002848:Chromatography, DEAE-Cellulose; D004262:DNA Restriction Enzymes / Q000494:pharmacology; D004279:DNA, Viral / Q000302:isolation & purification*; D004586:Electrophoresis
D004279:DNA, Viral; D004262:DNA Restriction Enzymes