Isolation of genomic DNA (Heiner Mueller)

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

Supplies & Equipment (autoclaved):

• Sterile scalpel or razor blade
• 50 ml tubes, centrifuge for 50 ml tubes
• Homogenizer
• Hybridization bottles and hybridization oven at 37°C and 50-55°C
• Dialysis tubing and clamps
• large (5-10 liter) beaker
• magnetic stirrer
• wet ice
• Pasteur pipettes

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. Collect the tissue (preferrably liver) as fresh as possible into 50 ml tube. Store on ice. Using a sterile scalpel or razor blade, cut the tissue into small pieces (mince). For later processing, the tissue can be frozen at -80°C or in liquid nitrogen. From 1 g of tissue ~ 2 mg of DNA can be expected.
   If working with liver, the gallbladder should be removed, because it contains high levels of degradative enzymes.
2. Prewarm 28.25 ml STE buffer to 50 - 55 °C. Add 1.25 ml 20% SDS (final conc. in 50 ml = 0.5%) and 0.5 ml of Proteinase K (final conc.in 50 ml = 100 µg/ml).
   To 1-10 g of minced tissue in a 50 ml tube add ice-cold STE buffer to a final volume of 20 ml. Homogenize tissue quickly. Add the prewarmed STE /SDS/Proteinase K solution.
3. Place tube in hybridization bottle and incubate rotating in hybridization oven at 55°C for 3-16 h (overnight).
4. Split sample evenly into two tubes. Allow to cool to room temperature. Extract protein with phenol: Add 10 ml phenol to each 25 ml sample; gently mix by inverting until well dispersed. Place on rocker to mix for one hour. After that, most of the dark brown protein should be in the phenol phase. Chill in ice for 10 min. Spin at 2500 rpm for 10 minutes. 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 brown color. After the last extraction step, centrifuge again for 10 min and remove the remaining trace of phenol at the tip of the tube.
6. Transfer the DNA solution to the dialysis tubing. Dialyze the DNA solution against 1 x TE buffer first at room temperature to avoid SDS precipitation (3 x 3 hours), and then at 4°C for 2 - 3 days with several changes of buffer (until A₂₇₀ of the dialysate is less than 0.05). Use large beaker (5-10 liter), stir.
7. Transfer the dialyzed DNA into a 50 ml tube. Add RNAse A to a final conc. of 100 µg/ml. Place in hybridization bottle and incubate rotating in hybridization oven at 37°C for 2 h.
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 3 - 16 hours (overnight).
9. Extract the DNA twice with phenol as described in steps #4-5.
10. Mix well with 0.1 vol. of 2 M NaOAc, then add 2 volumes of 100% ethanol at room temperature. Mix gently but thoroughly by inverting the tube several times. Long white threads of DNA will precipitate and conglomerate. Fish the DNA with a clean Pasteur pipette (end sealed and formed into a hook over Bunsen-burner). Shortly tip DNA on Kim-wipe to draw out ethanol. Do not over-dry the DNA or it will be difficult to redissolve.
11. Redissolve the DNA pellet in 10 ml 10mM Tris buffer pH 7.5 at 4°C overnight. If necessary, heat the DNA up to 37°C to help disolving the DNA. Add more Tris buffer if the DNA pellet is too large to be dissolved in 10 ml.
12. Dialyze the resulting DNA solution against 0.1 x TE buffer at 4°C for one week with several changes of buffer to remove trace of ethanol or phenol.
13. Determine the DNA concentration by reading absorbance at 260 nm of a diluted aliquot in TE buffer (1 absorbance unit = 50 µg/ml).
14. Store genomic DNA at 4°C.

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