Culture Volume: 100-250 mL

Prepare phase lock tubes, (spin 5' at 1500 rpm, RT)

1. Resuspend pellet in 12 mL of AE buffer. (50mM NaOAc pH 5.2, 10mM EDTA)
2. Add 960 uL of 20% SDS. Vortex.
3. Add 12 mL of AE saturated phenol, unbuffered (pH ~4.3)
4. Incubate at 65C, 10 minutes. Vortex every minute.
5. Spin 10000 rpm, in a Beckman JA-20, 15'
6. Collect the aqueous phase in a phase lock tube
7. add 13 ml chloroform, invert several times to mix.
8. Spin 3000 rpm, 10'
9. pour supernatant into new Rnase free Nalgene Oak Ridge tube
10. add 1/10th volume 3M NaOAc, ph 5.2.
11. add equal volume isopropanol.
12. spin 40 minutes 12,000 RPM in SS34.
13. Wash pellet with 10 ml 70% ethanol
14. Spin 12,000 RPM 20'
15. Dry pellet under vacuum 15'
16. Re-suspend pellet in 500 ul water or 5 mM Tris, pH 7.5
17. Save a small aliquot for quantification in a spectrophotometer at 260 nm, and run 10 ug on a gel to check integrity.

notes
This protocol uses unbuffered phenol. Unbuffered phenol is acidic and comes either as a solid (which you can melt at 65 C and aliquot) or as a saturated liquid. Once phenol has been exposed to an aqueous liquid it absorbs water and does not re-crystalize at room temperature.

Rinse oak ridge tubes with chloroform prior to use.