The white blood cells (Nucleated) (WBC) of peripheral blood are usually the most convenient source of human genomic DNA isolation. Human blood cell membrane is lysed by detergent Triton X-100, and mass of nucleus from cell population is collected by removing cytoplasmic contents (largely proteins) in the supernatant. Nuclear membrane is then disrupted by stronger detergent, SDS to release chromosome in lysate. Treatment with Proteinase K and Phenol: Chloroform can dissociate histones and other proteins from DNA and precipitate them. Free DNA is recovered in aqueous solution after centrifugation and concentrated by ethanol.
Requirements:
- Fresh blood (human)
- Lysis buffer A (0.32 M sucrose, 10 mM Tris-HCl, 5 mM MgCl2, 0.75% Triton X-100, pH 7.6, Sterilize buffer and add Triton)
- Lysis buffer B (sterile 20 mM Tris-HCl, 4 mM Na2-EDTA and 100 mM NaCl with pH 7.4)
- SDS (10%)
- Proteinase K (4 mg/ml)
- RNase (5 mg/ml)
- Phenol: Chloroform (1:1)
- Chloroform: Isoamyl alcohol (24:1)
- Sodium acetate (3 M)
- Abs. Ethanol / Isopropanol
- TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
Procedure:
- Add 2 ml of whole blood in a beaker containing 8 ml of Lysis Buffer A. Mix well and keep at 4 o C (on ice) for 10-15 minutes.
- Take 1.5 ml of the lysed blood to a microfuge tube and centrifuge at 4°C at 1500 rpm for 10 min. Decant the supernatant, collecting the pink pellet of nuclei.
- Add 500 μl of cold lysis buffer B to the pelleted and mixed gently.
- Add 1/10 volume (50 μl) of 10% SDS and swirl to mix.
- Add 1/100 volume of Proteinase K (4 mg/ml) and incubate at 37°C for 30 minutes with gentle shaking.
- Add 2 μl RNase and mix gently and hold for 10 min.
- Add an equal volume of phenol:chloroform (1:1) and mix well to form an emulsion. Centrifuging at 8000 rpm for 10 min.
- Carefully decant the aqueous (top) phase to a clean microfuge tube.
- Add an equal volume of chloroform:isoamyl alcohol (24:1) and mix gently. Centrifuging at 8000 rpm for 5 min.
- Decant the aqueous phase to a clean microfuge tube and add 1/10 volume of 3 M sodium acetate.
- Add 1 volume of isopropanol and repeatedly invert the tube until a light precipitate forms. Centrifuge at 13,000 rpm for 15 min.
- Decant off the supernatant as completely as possible and dissolve the pellet in 100 μl of TE buffer.
- Store the analyte in deep freeze (-20°C).
Recomended concentration of Agarose for Gel electrophoresis- 0.8%.
References
Tiwari, K. B. & Ghimire, P., 2010. A Practical handbook for Microbial Genetics & Molecular Biology. 1st ed. s.l.:Kantipur College of Medical Science
