Extraction of DNA from E.

coli

Reagents to be prepared

1. STE Buffer

2. TE buffer
3. Phenol
4. chloroform Isoamyl alcohol (24:1)

1. To extract the DNA from Gram-negative (E.coli) 1 ml cell suspension was centrifuged at 10,000
rpm for 3 min to pellet the cells.
2. After removing the supernatant, the cells were washed twice with 400 µl STE Buffer (100 mm
nacl, 10 mm Tris/hcl (invitrogen), 1 mm EDTA (invitrogen), ph 8.0).
3. Then the cells were centrifuged at 10,000g for 3 min,
4. The pellets were resuspended in 200 µl TE buffer (10 mmtris/hcl, 1 mm EDTA, ph 8.0).
5. Then 100µl Tris-saturated phenol (ph 8.0) was added to these tubes, followed by a vortex-
mixing step of 60 s for bacteria, to lyse the cells,
6. The samples were subsequently centrifuged at 13,000 rpm for 5 min at 4 0C to separate the
aqueous phase from the organic phase.
7. Then 160µl upper aqueous phase was transferred to a clean 1.5 ml tube.
8. 40µl TE buffer was added to make 200µl and mixed with 100µl chloroform Isoamyl alcohol
(24:1) and
9. Centrifuged for 5 min at 13, 000g at 4 0C. (Lysate was purified by chloroform Isoamyl alcohol
(24:1) extraction until a white interface was no longer present)
10. This procedure might have to be repeated two to three times.

11. 160µl upper aqueous phase was transferred to a clean 1.5 ml tube.

12. The aqueous phase contained purified DNA and was directly used for the subsequent

experiments or stored at -20 0C.