DNA extraction protocol Phenol/Chloroform/Isoamyl alcohol

1. Start with 1-5 milion cells (depending on sample volume and total number of cells)
2. Centrifuge cells 5 min at 3.000 rpm and discard supernatant (PBS after washing)
3. Add 400 µL SNET lysis buffer (20 mM Tris-Cl (pH 8.0) 5 mM EDTA (pH 8.0) 400
mM NaCl 1% (w/v) SDS; Molecular Cloning – a laboratory manual, Maniatis, 3rd ed.,
p.6.24) and Proteinase K stock solution to 400 µg/ml). Mix by pipetting.
4. Add one volume of phenol:chloroform:isoamyl alcohol (25:24:1) (Ambion, Thermo
fisher) to sample, and shake by hand thoroughly for approximately 20 seconds (milky
emulsion should form).
5. Centrifuge at room temperature for 10 minutes at 10.000 rpm. Remove the upper
aqueous phase, and transfer the layer to a fresh tube.
6. Add one volume of chloroform:isoamyl alcohol (24:1) and shake by hand thoroughly
for approximately 20 seconds.
7. Centrifuge at room temperature for 10 minutes at 10.000 rpm. Remove the upper
aqueous phase, and transfer the layer to a fresh tube.
8. Add one volume of chloroform:isoamyl alcohol (24:1) and shake by hand thoroughly
for approximately 20 seconds.
9. Centrifuge at room temperature for 10 minutes at 10.000 rpm. Remove the upper
aqueous phase, and transfer the layer to a fresh tube.
10. Add 0,1 volume of 2M NaCl to aqueous phase and 2 volumes of cold 96% ethanol and
mix tube by inverting. Refrigerate for 30 min to one hour for better precipitation.
11. Centrifuge at +4°C for 30 minutes at maximum speed (15.000 rpm).
12. Discard supernatant and wash pellet with 750 µL 75% ethanol.
13. Discard supernatant and air dry for 5 min.
14. Re-suspend in appropriate volume of elution buffer (based on input amount) (in our
case 150 µL of aqua proinjectione, ph.jug.4)