Mr. Khadga Bikram Angbuhang Faculty: Department of Microbiology GoldenGate Intl College Research officer: LEAD Nepal.
Genomic DNA extraction from animal tissues Principle: The isolation of DNA usually begins with lysis of tissue or cells. Lysis is carried out in a salt solution, containing detergents (SDS) and proteases (Proteinase K). SDS solubilizes the cell membrane and lipid membranes of internal organelles and denatures proteins (enzymes) which release chromosome in lysate. Treatment with Proteinase K and Phenol: Chloroform can dissociate histones and other proteins from DNA and precipitate them. Tris-HCL is a buffer, which retain constant pH; ethylenediaminetetraacetic acid (EDTA), binds metal ions. Chloroform solubilizes lipids and a lot of proteins and separate proteins and polysaccharides from nucleic acids in the cell. Chloroform is denser than water solutions and thus after spinning, Chloroform and water will separate into two distinct phases. The lower phase will be Chloroform. This is the phase that proteins and polysaccharides find most chemically attractive. The upper aqueous phase will contain your DNA. Free DNA is recovered in aqueous solution after centrifugation and concentrated by ethanol. DNA is less soluble in solutions containing isopropanol than in solutions containing ethanol. Precipitation with isopropanol is performed at room temperature to lessen the risk that solutes like sucrose or sodium chloride will be co precipitated with the DNA. DNA does NOT dissolve in ethanol (Chilled Ethanol). Cold alcohol is used to separate DNA out of water-based solutions. Material Required:
1. 2. 3. 4. 5. 6. 7. 8. 9.
Extraction buffer: (NaCl 10mM; Tris-HCl 10mM; EDTA 10mM, pH 8.0) SDS - 10%. 3 M Sodium acetate, pH 4.8 by acetic acid Phenol: Chloroform (1:1) Chloroform: isoamyl alcohol (24:1) RNase A: (1 mg/ml in 5 mM Tris-HC1, pH 8.0) Proteinase K: 10 mg/ml stock 70% ethanol TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0; Autoclave before use.
1. Transfer about 10-20 mg animal tissue to an MFT 1.5mL tube labeled with an 2. 3. 4. 5. 6. 7. 8.
identification number. Add liquid nitrogen and grind the tissue for 1 minute with a pestle. Add 300μl Extraction buffer and 100μl SDS 5%. Add 15μl Proteinase K. Grind the tissue with a pestel. Incubate the tube at 60°C for at least 30 minutes. Add 1-2 µl of RNase and incubate for 10 min at RT. Add equal vol. of Phenol: Chloroform (1:1), mix gently and keep for 10 min at RT.
9. Centrifuge at 8,000 rpm for 15 min. 10. Carefully decant the aqueous (top) phase to a clean microfuge tube. 11. Add an equal volume of chloroform: isoamyl alcohol (24:1) and mix gently and Centrifuging at 8000 rpm for 5 min. 12. Decant the aqueous phase to a clean microfuge tube and add 1/10 or equal volume of 3 M sodium acetate and 300μl 10 or equal volume of isopropanol. 13. Mix gently inverting the MFT. 14. Leave in cold temperature for 30 minutes. 15. Centrifuge at 13,000 rpm for 5 minutes. 16. Eliminate the supernatant and collect pellet (in same MFT). 17. Wash pellet adding 500μl ethanol 70%. 18. Centrifuge at 13,000 rpm for 5 minutes. 19. Eliminate the supernatant. 20. Dry the pellet for 10-15 minutes at 60°C to evaporate remaining ethanol. 21. Resuspend pellet in 50μl TE.