DNA purification by polycarbonate filters.
Organic solvent free procedures are described for the purification of mammalian DNA from rat liver, kidney, spleen, lung and brain. The basis of the purification procedures are the use of the detergent sodium dodecyl sulfate (SDS) and inert polycarbonate filters with 2 mcm pores which retain long macromolecules such as DNA while allowing other cellular constituents to pass through the pores. Neither proteinase K nor use of peristaltic pumps were found to be absolutely required for DNA purification, although it is desirable to use peristaltic pumps to control solution flow rates. Of the 12 different procedures (1.2-4.2 h required) evaluated, purified mammalian DNA contained from 0 detectable to 1.5% of the original protein remaining as a contaminant. With the 4 recommended procedures, the average of 8 experimental values of DNA to protein ratios was 1.81. Depending on the diameter of the individual filter, about 12-90 mcg of rat liver DNA can be purified from each separate tissue or cell sample.
Kitchin
K T
KT
Genetic Toxicology Division, US Environmental Protection Agency, Research Triangle Park, NC.
Brown
J L
JL
D000818:Animals; D002021:Buffers; D002417:Cattle; D004247:DNA / Q000302:isolation & purification*; D019286:Endopeptidase K; D005069:Evaluation Studies as Topic; D005260:Female; D005374:Filtration; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000737:chemistry; D011076:Polycarboxylate Cement; D051381:Rats; D017207:Rats, Sprague-Dawley; D012697:Serine Endopeptidases; D012967:Sodium Dodecyl Sulfate
D002021:Buffers; D011076:Polycarboxylate Cement; C005506:polycarbonate; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D012697:Serine Endopeptidases; D019286:Endopeptidase K