A bacteriophage lambda DNA purification procedure suitable for the analysis of DNA from either large or multiple small lysates.
A method for the efficient preparation of high quality bacteriophage lambda DNA from cleared lysates is described. Advantages of the method include high DNA yields (typically around 0.8 micrograms of DNA/1 ml of cleared lysate), speed of processing (approximately 2 h from lysate to DNA), economy, and the absence of any requirement for phenol or chloroform extractions. The technique involves the concentration of phage particles by standard polyethylene glycol precipitation followed by enzymatic treatment to remove contaminating RNA and DNA. Phage particles are then lysed with sodium dodecyl sulfate (SDS) at elevated pH and temperature. Contaminating protein/SDS complexes are rendered insoluble by the addition of potassium acetate and removed by centrifugation. The quality of the resultant DNA is comparable to that prepared by cesium chloride banding for all standard molecular biological purposes providing that spermidine is included in all restriction endonucleases digestions.
Lockett
T J
TJ
CSIRO Division of Biotechnology, Laboratory for Molecular Biology, North Ryde, New South Wales, Australia.
D000085:Acetates; D019342:Acetic Acid; D010582:Bacteriophage lambda / Q000032:analysis*; D011232:Chemical Precipitation; D004279:DNA, Viral / Q000302:isolation & purification*; D004720:Endonucleases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008722:Methods; D011092:Polyethylene Glycols; D012967:Sodium Dodecyl Sulfate; D013696:Temperature
D000085:Acetates; D004279:DNA, Viral; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D004720:Endonucleases; D019342:Acetic Acid