The Isolation of High Molecular Weight Eukaryotic DNA: C. G. P. Mathew

Chapter 5
The Isolation Molecular Eukaryotic

C. G. P. Mathew
MRC Molecular and Cellular Cardiology Research Unit, University of Stellenbosch Medical School, Tygerberg, South Africa
of High Weight DNA
Introduction
The isolation of high molecular weight eukaryotic DNA m good yield is an Important prerequisite for the analysis of specific sequences by Southern blotting (Chapter 9), or for molecular cloning m phage or cosmld vectors (Chapter 49). In the procedure described below, cells from the organism are disrupted by homogemzatlon m Trlton X-100 and the nuclei pelleted by centrifugation The nuclei are then resuspended and treated with SDS, which dissociates the DNA-protem complex. The protein IS removed by dlgestion with a proteolytlc enzyme, protemase K, and phenol extractlon Finally, the nucleic acids in the aqueous phase of the extract are treated with rlbonuclease and the DNA 1s precipitated with ethanol. 31
32
Mathew
Whole blood IS a convenient source of DNA from larger mammals, but the procedure can easily be modified to isolate DNA from any cellular tissue
Materials
1. Cell lysrs buffer: 320 mM sucrose, 1% (v/v) triton X-100; 5 mM M&l,; 10 mM Tris-HCl, pH 7.6, 2. Saline-EDTA: 25 mM EDTA (pH 8.0); 75 mM NaCl. 3. Sodium dodecyl sulfate: prepare a 10% (w/v) stock solution. 4 Protemase K prepare a 10 mg/mL stock solution 5 5M sodium perchlorate. 6. Phenol-chloroform* high-quality commercial phenol can be used without redistillation Batches that are pmk or yellow should be redistilled at 160C to remove contaminants. Melt the phenol at 68C, and add 8hydroxyquinolme (antioxidant) to a final concentration of 0.1% Mix with an equal volume of chloroform, and extract several times with O.lM Tris-HCl, pH 8 0 7. Chloroform* isoamyl alcohol (24 1). 8. TE buffer: 10 mM Tris-HCl (pH 7 5); 1 mM EDTA. 9. 20 x SSC: 3.OM NaCl, 0.3M sodium citrate, pH 7.0 10. Ribonuclease: dissolve pancreatic ribonuclease A at 5 mg/mL m 10 mM Tris-HCl, pH 7 5. Heat at 80C for 10 min. 11. Ethanol. 70% (v/v) and absolute. Store solutions 1 and 6 at 4C, and 4, 10, and 11 at -20C. All other solutions may be stored at room temperature.
Method
The procedure given from whole blood It can DNA from cultured cells The method 1s based al (1) below is for isolation of DNA be modified slightly to prepare or tissues (see Note 1) on that described by Kunkel et
1. Collect 10 mL of whole blood in tubes containing EDTA or citrate as anticoagulant.
Isolation of Eukaryotlc
DNA
33
2. Add the blood to 60 mL of cell lysrs buffer at 4C and homogenize in a Potter homogenizer with a loosefrttmg pestle Pellet the nuclei by centrifugatron at 2500g for 20 mm at 4C 3. Suspend the pellet m 8 mL of salme-EDTA, and add 0.8 mL of 10% (w/v) SDS. Vortex briefly. 4. Add 50 PL of the proteinase K solution and incubate at 37C for 24 h. 5. Add 0.5 mL of 5M sodium perchlorate and 8 mL of phenol-chloroform. Mix gently until homogeneous, and separate the phases by centrifugation at 12,OOOg for 10 mm at 10C. 6. Remove the aqueous phase wrth a wide-bore prpet, and extract rt with an equal volume of chloroform-rsoamyl alcohol. MIX gently, and separate the phases as m step 5. 7. Precipitate the DNA from the aqueous phase by adding 2 vol of cold absolute ethanol (seeNote 2) Lift out the precipitate wrth the sealed end of a Pasteur pipet, and shake mto 1 mL of TE buffer. Dissolve overnight at 4C, with gentle mrxmg 8. Add 100 PL of 20 X SSC and 10 PL of rrbonuclease, and incubate for 1 h at 37C 9. Add 2 mL of sterile distilled H20, and extract the solution twice with chloroform-rsoamyl alcohol 10. Precrprtate the DNA by adding 2 vol. of absolute ethanol, and centrifuge at 5000g for 5 mm at 5C. Wash the pellet with 70% ethanol and dry under a vacuum Drssolve the DNA m 0.5 mL of sterile drstrlled H20. 11. Scan a dilution of the DNA from 220 to 300 nm (see Note 3). Determine the absorbance at 260 nm and calculate the DNA concentration by assummg that the Apcm,260 1s 200 [i.e., a 1 g/100 mL solution in a l-cm light path has an absorbance of 200 at 260 nm (2)] The yield of DNA from 10 mL of human blood IS
200400 /.Lg.
Notes
1 DNA can be isolated from cultured cells as follows suspend cell pellet m tissue culture medium at a concentration of lo7 cells/ml Add 5 vol of cell lysrs buffer
34
Mathew and homogenize (Step 2). Contmue with the procedure described for whole blood, but scale the volumes up or down according to the volume of cell lysis buffer used. To isolate DNA from tissues, mince the tissue and blend it in liquid nitrogen, using a stainless-steel Waring blendor. Let the liquid nitrogen evaporate and add the powder to approximately 10 vol of lysis solution (3). After pelleting the nuclei (Step 2), follow steps 3-11 as described in the Method section. 2. In order to prepare DNA of very high mw ( > 30 kb), mixmg with phenol and chloroform should be done very gently, and the DNA should not be ethanolprecipitated (3). Substitute precipitation steps 7 and 10 with extensive dialysis against several changes of TE buffer. In this case, the final product should not migrate faster than intact A-DNA on a 0.4% agarose gel. 3. The scan of the DNA will detect impurities such as protein contammation, which can be removed by repeatmg the phenol-chloroform extraction, or traces of phenol, that will be removed by repeated extractions with chloroform-isoamyl alcohol. 4. Glassware and plasticware, such as Eppendorf tubes and automatic pipet tips, should be sterilized by autoclavmg.
References
1 Kunkel, L. M , Smith, K D., Boyer, S H., Borgaonkar, D S., Wachtel, S S , Miller, 0 J , Breg, W R , Jones, H W., and Rory, J. M. (1977) Analysis of human Y-chromosomespecific reiterated DNA m chromosome variants Proc Nut1 Acad Scl USA 74, 1245-1249 2 Old, J M , and Higgs, D. R (1983) Gene Analysis In Methods m Hematology The Thalassaemzas (ed. Weatherall, D J ), p. 78 Butler & Tanner, Rome and London 3 Mamatis, T , Fritsch, E F., and Sambrook, J. (1982) Molecular Clonmg A laboratory manual Cold Sprmg Harbor Laboratory, New York

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Chapter 5

The Isolation Molecular Eukaryotic

of High Weight DNA

1. Collect 10 mL of whole blood in tubes containing EDTA or citrate as anticoagulant.

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