A simple salting out procedure for extracting DNA from human nucleated cells.

SA Miller, DD Dykes, HF Polesky - Nucleic acids research, 1988 - ncbi.nlm.nih.gov
SA Miller, DD Dykes, HF Polesky
Nucleic acids research, 1988ncbi.nlm.nih.gov
One of the obstacles encountered when extracting DNA from a large number of samples is
the cumbersome method of deproteinizing cell digests with the hazardous organic solvents
phenol and isochloroform. Several other non-toxic extraction procedures have been
published, but require either extensive dialysis (1) or the use of filters (2). A rapid, safe and
inexpensive method was developed to simplify the deprotein-ization procedure. This method
involves salting out of the cellular proteins by dehydration and precipitation with a saturated …
One of the obstacles encountered when extracting DNA from a large number of samples is the cumbersome method of deproteinizing cell digests with the hazardous organic solvents phenol and isochloroform. Several other non-toxic extraction procedures have been published, but require either extensive dialysis (1) or the use of filters (2). A rapid, safe and inexpensive method was developed to simplify the deprotein-ization procedure. This method involves salting out of the cellular proteins by dehydration and precipitation with a saturated NaCl solution. Buffy coats of nucleated cells obtained from anticoagulated blood (ACD or EDTA) were resuspended in 15 ml polypropylene centrifugation tubes with 3 ml of nuclei lysis buffer (10 mM Tris-HCl, 400 mM NaCl and 2 mM Na2EDTA, pH 8.2). The cell lysates were digested overnight at 37 C with 0.2 ml of 10% SDS and 0.5 ml of a protease K solution (1 mg protease K in 1% SDS and 2 mM Na2EDTA). After digestion was complete, 1 ml of saturated NaCl (approximately 6M) was added to each tube and shaken vigorously for 15 seconds, followed by centrifugation at 2500 rpm for 15 minutes. The precipitated protein pellet was left at the bottom of the tube and the supernatant containing the DNA was transferred to another 15 ml polypropylene tube. Exactly 2 volumes of room temperature absolute ethanol was added and the tubes inverted several times until the DNA precipitated. The precipitated DNA strands were removed with a plastic spatula or pipette andtransferred to a 1.5 ml microcentrifuge tube containing 100-200 pl TE buffer (10 mM Tris-HCl, 0.2 mM Na2EDTA, pH 7.5). The DNA was allowed to dissolve 2 hours at 37 C before quantitating. The DNA obtained from this simple technique yielded quantities comparable to thoseobtained from phenol-chloroform extractions. The 260/280 ratios were consistently 1.8-2.0, demonstrating good deproteinization. Restrictions were performed using a number of different enzymes requiring high, medium or low salt concentrations, all resulting in complete restriction. This procedure has been used inour laboratory on several thousand blood samples for parentage, population and forensic studies. This technique is used with ournon-isotopic hybridizationprocedures (3) rendering the entire process of RFLP analysis free of toxic materials.
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