April 24, 1990
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.
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.
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.
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.
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.
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.