Isolation of genomic DNA (Simon Kwok)

All glassware should be autoclaved and reagents should be autoclaved or sterile filtered. 

Supplies & Equipment (autoclaved):

• 50 ml tubes, centrifuge for 50 ml tubes
• mortar and pestle
• 250 ml Erlenmeyer flasks
• 125 ml Erlenmeyer flasks and shaker / incubator at 50-55°C or:
• Hybridization bottles and hybridization oven at 50-55°C
• Dialysis tubing and clamp
• dry ice
• wet ice
• 37°C waterbath
• Speed Vac

Reagents:

• STE buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 100 mM EDTA)
• TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA)
• 20 % SDS (20 g SDS per 100 ml, sterile filtered)
• 2 M NaOAc (sodium acetate), pH 5.2 = dilute from 3 M stock, store at 4°C
• Phenol = phenol : chloroform : isoamyl alcohol (20 : 19.2 : 0.8, v/v), saturated with STE buffer
• Proteinase K = 10 mg/ml in 10 mM Tris-HCl pH 7.5, freshly prepared
  • 10 mg/ml = 100 x, i.e. per 50 ml sample will need 5 mg in 500 µl
• RNAse A = 10 mg/ml in 10 mM Tris-HCl pH 7.5, boil for 10 min. store at -20°C
  • 10 mg/ml = 100 x, i.e. per 50 ml sample will need 5 mg in 500 µl

Procedure:

1. For blood samples, centrifuge 50 - 100 ml heparinized blood at 2500 rpm for 15 min. Remove plasma. Collect the white buffy coat (leucocytes) with a spatula. Keep frozen on dry ice.
2. For organs, cut a small piece of the tissue (about 1 gram) with a razor blade. Grind it to a fine powder with dry ice using a mortar and pestle. Keep tissue powder frozen on dry ice.
3. This step either in 125 mL Erlenmeyer flask and shaker incubator or hybridization bottle and oven at 55°C. Add 1.25 ml of 20% SDS to 50 ml of STE buffer in a 125 ml flask (final [SDS] = 0.5%), prewarm to 50 - 55 °C. Then add 0.5 ml of Proteinase K (final conc. = 100 µg/ml), followed by the buffy coat or tissue / dry ice powder. Mix by gentle swirling. [Note: The tissue / dry ice powder should be added in slowly until no more can be solubilized.] Incubate at 50 - 55°C for 3 - 16 hours with gentle swirling.
4. Allow to cool to room temperature. Transfer the digest to a 250 ml flask. Extract with equal volume (50 ml) of phenol at room temperature for 10 min. by gentle swirling (extraction in large flasks is to increase surface contact between the aqueous and phenolic phases). Chill in ice for 10 min. Remove the phenol (bottom phase) with a 10 ml pipet. Do not pipet the DNA solution or you will break the DNA!
5. Repeat step #4 twice until there is no more red color (~2-3 times). After the last extraction step, transfer the DNA solution to a 50 ml tube, centrifuge for 15 min. Remove the trace of phenol at the tip of the tube with a pasteur pipet.
6. Transfer the DNA solution to the dialysis tubing by pouring through a sterile funnel. Dialyze the DNA solution against TE buffer first at room temperature to avoid SDS precipitation, and then at 4°C for 2 - 3 days with several changes of buffer (until A₂₇₀ of the dialysate is less than 0.05).
7. Transfer the dialyzed DNA to a 125 ml Erlenmeyer flask. Add RNAse A to a final conc. of 100 µg/ml. Incubate at 37°C for 2 hours in shaker / incubator.
8. Add SDS to a final conc. of 0.5%, EDTA to 25 mM, and proteinase K to 100 µg/ml. Incubate the mixture at 50-55°C for 2 hours (or overnight if necessary).
9. Extract the DNA twice with phenol as described in steps #4-5.
10. Transfer the DNA solution to a 250 ml flask. Mix with 0.1 vol. of 2 M NaOAc and 2 vol. of ethanol at room temperature.
11. Collect DNA precipitate immediately. Rinse once with 70% ethanol. Dry in Speed Vac briefly (5 min.). Do not over-dry the DNA or it will be difficult to redissolve.
12. Redissolve the DNA pellet in 2-5 ml TE buffer at 4°C overnight. If necessary, heat the DNA up to 37°C to help disolving the DNA.
13. Dialyze the resulting DNA solution against 0.1 x TE buffer at 4°C with several changes of buffer to remove trace of ethanol or phenol.
14. Determine the DNA concentration by reading absorbance at 260 nm of a diluted aliquot in TE buffer (1 absorbance unit = 50 µg/ml).

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