A method for quantitative recovery of DNA from plate lysates of bacteriophage lambda-derived clones.
A method is described for the purification of DNA from recombinant clones in lambda vectors. Plate lysates are used as the starting material, with the phage suspension being either loaded directly onto mini-cesium chloride two-step gradients or first subjected to precipitation with polyethylene glycol-8000 before application to the gradients. A single centrifugation step is used and the DNA is isolated from the harvested bacteriophage by incubation at room temperature with formamide followed by ethanol precipitation. The small-volume gradients were effective in isolating phage particles in the range of 1.0 to 12.5 x 10(11) pfu with quantitative recovery of approximately 7 micrograms DNA/10(11) pfu. The method was tested with a cDNA clone in lambda gt10 (as an example of a lambda-derived vector which gives a plate lysate with a high titer) and rat genomic fragments cloned into EMBL3A (for which the plate lysates are of low titer). For both vectors, the purified DNA appeared free of inhibitory contaminants as assessed by restriction endonuclease digestion and subcloning of fragments.
Mitchell
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Lipoprotein-Atherosclerosis Unit, Baker Medical Research Institute, Prahran, Victoria, Australia.
D010582:Bacteriophage lambda / Q000235:genetics*; D016923:Cell Death / Q000502:physiology; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis*; D004926:Escherichia coli; D005822:Genetic Vectors; D016679:Genome, Viral; D015203:Reproducibility of Results; D017186:Titrimetry
D004279:DNA, Viral