Method: Salting Out Procedure for Human DNA Extraction

April 24, 1990

C. Helms

Principle:

This procedure avoids using phenol and chloroform by using high salt concentrations to remove proteins. It is rapid, safe and inexpensive. The procedure is written for the quantity of white blood cells obtained from one or two tubes of blood, and should be considered a miniprep for human DNA. Average yields are similar to that obtained with the phenol-chloroform extraction procedure (50- 200 ug), and the quality of DNA is excellent. The procedure may be scaled up to handle larger samples.

Time required:

2 days

Special solutions:

• Saturated NaCl solution

Procedure:

Day 1

1. Isolate nuclei from 1-2 tubes of blood (collected in ACD or EDTA tubes):

   Add 9 volumes Buffer A, mix well and hold on ice 2 minutes. Spin at 1500 rpm at 4deg.C for 15 minutes. Resuspend the nuclei pellet in 5 ml Buffer B and transfer to a 15 ml polypropylene centrifuge tube.

2. Add 500 ul 10% SDS and 55 ul proteinase K (10 mg/ml stock). Incubate at 37deg.C overnight on a low-speed rocker or orbital shaker.

1. Add 1.4 ml saturated NaCl soution (approximately 6M) to each tube. Shake vigorously for 15 seconds. Spin the tubes at 2500 rpm in the Beckman low speed centrifuge for 15 minutes.
2. Transfer the supernatant to another 15 ml polypropylene tube, leaving behind the precipitated protein pellet.
3. Add exactly two volumes of room temperature 100% ethanol and invert the tube several times until the DNA precipitate is visible.
4. Remove the DNA strands with a plastic spatula or pipet tip and transfer to an eppendorf tube containing 100 - 200 ul TE. Allow the DNA to dissolve at least 2 hours at 37deg.C before quantitating.

Solutions:

• Buffer A

   0.32 M sucrose             109.5 g sucrose 10 mM Tris HCl pH 7.6      10 ml 1 M Tris-HCl pH 7.6 5 mM MgCl2                 5 ml 1M MgCl2 1 % Triton-X-100  bring volume to 1 liter with deionized water Sterilize above solution by autoclaving, then add 10 ml Triton-X-100.

• Buffer B

   25 mM EDTA pH 8.0         50 ml EDTA pH 8.0 75 mM NaCl                40 ml  5 M NaCl  Bring volume to 1 liter with deionized water. Sterilize by autoclaving.

• Saturated NaCl solution ( approximately 6M)

  Dissolve 35 grams of NaCl in a total volume of 100 ml (deionized water). If the solution has no precipitate, add 2 grams NaCl and mix. Repeat until no more NaCl will go into solution. Filter sterilize.

References:

S. A. Miller, Dykes, D. D., and H. F. Polesky . (1988). " A simple salting out procedure for extracting DNA from human nucleated cells" Nucleic Acids Research 16: 1215.