ISOLATION OF GENOMIC DNA

DNA Isolation from Tissue and Cell Lines:
(Modification of Blin & Stafford Nucl. Acids Res. 3:2303, 1976)

Prepare in advance: TE-saturated phenol, 55°C water bath, and a 55°C solution of 20 mM Tris-HCl, pH 7.8; 50 mM EDTA, 1% SDS.

Day 1
  1. Weigh out 1-2 g of frozen liver. (This tissue should have been chopped into approximately ¼-½" pieces after dissection and quick frozen by immersion in liquid nitrogen. These tissue sections can be stored at -70°C. Mam malian tissue culture cells may also be used. There are about 108 cells per packed ml. This will give a comparable amount of DNA to the liver protocol. This protocol yields 5-10 mg of DNA.)
  2. Transfer to mortar and grind with a pestle to a granular appearance under liquid nitrogen.
  3. Add proteinase-K to 100µg/ml in above solution at 55°C. Add liver granules slowly to the solution while mixing to avoid clumping of t he granules. A tall, wide-mouth beaker may be better than a narrow-mouth Erlenmeyer flask to avoid clumping in one area. Incubate while gently swirling at 55°C for 3 hours.
  4. Extract 3 times with 100 ml of phenol. Centrifuge at 1800xg (3000 rpm in Beckman TJ-6) for 1 minute to separate the phases in 250 ml bottles. Extract the aqueous layer with sec-butanol to condense the DNA to about ½ of the original volume.
  5. Extract with ether 3 times. Blow off ether with N2 gas.
Day 2
  1. Bring the DNA solution to 10 mM NaCl.
  2. Add heat-treated RNAse A to 100µg/ml. Gently swirl at 37°C for 3 hours.
  3. Extract twice with phenol, concentrate 5-10 fold with sec-butanol to a few mls, and extract twice with ether. Centrifugation after sec-butanol extractions helps to separate the phases and perhaps results in higher yields.
  4. Dialyze at 4°C in freshly boiled dialys is tubing against cold 20 mM Tris-HCl, pH 7.8, 10 mM NaCl, 0.2 M EDTA. Dialyze 3x against 2 liters of solution.
  5. Quantitate DNA with a spectrophotometer at 260 nm and 280 nm. A typical yield from 1 g of liver or 108 cells is 5-10 mg with a 260/280 ratio of 1.7-1.8.

Isolation from Peripheral Blood Leukocytes:
Modification of Wyman and White PNAS 77:6754-6758 (1980)
  1. Start with 10-50 ml peripheral blo od in anticoagulant, EDTA is preferred. Centrifuge to pellet the cells at 300xg (1200 rpm in Beckman TJ-6 centrifuge) for 10 minutes at room temperature. Remove and freeze the plasma (if needed). Transfer buffy coat cells to another tube. Resuspend the remaining buffy coat cells and red blood cells in PBS with 0.1% EDTA (1:186 x dilution of 0.5 M EDTA stock) and respin. Add the resulting buffy coat cells to those initially collected, and spin once or twice to reduce red blood cells by transferring leuk ocytes to a new tube. A final pellet containing about equal volumes of white blood cells and red blood cells is fine.
  2. Transfer buffy coat cells and incubate them for 3 hours while gently rotating in 10-20 ml of a pre-heated 55°C lysis solution (1 mM EDTA, 10 mM Tris-HCl, pH 7.8, 10 mM NaCl, 1% SDS and 100µg/ml proteinase-K).
  3. Extract 3-4 times with a phenol/chloroform solution, discarding the lower layer each time. Ether or chloroform extract. You should not see hemoglobin af ter the final extraction.
  4. Ethanol precipitate. Resuspend in 1-2 ml of TE and treat with RNAse A @ 20 µg/ml for 1 hour, 37°C.
  5. Extract 3 times with phenol and the ether. Ethanol precipitate and resuspend in 0.5 ml of TE. A typical yield is 200-400 µg of DNA with a 260/280 ratio of 1.6 - 1.8.

RECIPES

20 ml Lysis Solution: (1 mM EDTA, 10 mM Tris-HCl, pH 7.8, 10 mM NaCl, 1% SDS and 100µg/ml proteinase-K). < DD>7.52 ml H2O
40 µl 0.5 M EDTA
40 µl 5 M NaCl
400 µl 0.5 M Tris-HCl, pH 7.8
2 ml 10% SDS
2 mg proteinase-K (added just before adding the cells)
TE (10 mM Tris-HCl pH 7.8; 1 mM EDTA)
For 600 ml solution:
0.727 g Tris
1.2 ml of 0.5M EDTA
pH to 7.8 with approximately 7 drops of concentrated HCl with a pasteur pi pet (ACID - wear rubber gloves), or about 4 ml of 1 M HCl. QS to 600 ml with water and autoclave.
RNAse (10 mg/ml)
Dissolve RNAse A in water. Boil for 5 minutes. Store at -20° C. Some protocols also add an equal volume of 40% glycerol and a half volume of 0.5 M NaCl.
    Send comments and updates to  Dr. Bart Frank, < /a> Arthritis and Immunology Program, OMRF
Return to Protocols: Table of Contents
Return to the Frank Lab Home Page
Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. I solation of Genomic DNA. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/genomdna.html). 1997. Oklahoma City. Revision Date: October 2, 1997."