- Pick a single colony from a plate and inculate 250ml of LB broth containing the appropriate antibiotic. Grow the cells overnight in a shaker at 37°C.
- Centrifuge the cells at 5,000g, 4°C for 15m.
- Resuspend the cell pellet in 6ml of ice-cold freshly prepared lysis buffer:
Lysis buffer
25mM Tris.HCl, pH8.0
10mM EDTA
50mM glucose
- Thoroughly resuspend the cells by pipetting them up and down with a 10ml pipette. Incubate in ice water for 10m.
- Add 12ml of freshly prepared 0.2M NaOH/1% SDS. Carefully mix by inversion and incubate in ice water for 10m. Do not vortex.
- Add 7.5ml of 3M sodium acetate, pH4.6. Carefully mix by inversion or gentle vortexing and incubate in ice water for 20m.
- Centrifuge at 12,000 x g for 15m. Transfer the supernatant to another tube and discard the precipitate.
Add DNAase-free RNAase A to a final concentration of 20µg/ml. Incubate at 37°C for 20m. - Extract twice with 1 volume of TE-saturated phenol/chloroform. Vortex for 1m and centrifuge at 12,000g for 5m.
- Transfer the upper, aqueous phase to a fresh tube and add 1 volume of chloroform:isoamyl alcohol (24:1), vortex for 1, and centrifuge at 12,000g for 5m.
- Transfer the upper, aqueous phase to a fresh tube and add 2 volumes of ethanol. Leave at -20°C for 30m, then centrifuge at 12,000 x g for 20m.
- PEG purify.
You can expect about 500μg from this protocol.
| < Prev | Next > |
|---|
