Plasmid Extraction

Protocol

1. The day before the experiment: Culture E. coli carrying plasmid of interest in a 50-ml centrifuge tube with 6.5 ml of LB + Cm (Chloramphenicol: 34 ug/ml), shaking at 37°C, overnight.

2. On the day: Centrifuge the E. coli at 15,000 xg for 5 min.

3. Discard the supernatants, add 200 ul of PD1 Buffer, mixing by vortex.

4. Add 200 ul of PD2 Buffer, mixing by gently swirling the tube. (Do NOT vortex)

5. Add 300 ul of PD3 Buffer, mixing by gently swirling the tube. (Do NOT vortex)

6. Centrifuge the lysates at 17,000 xg for 10 min.

7. Transfer the supernatants into the Column (Discard the pellet). Centrifuge at 15,000 xg for 30 sec.

8. Add 400 ul of W1 Buffer. Centrifuge at 15,000 xg for 30 sec.

9. Add 600 ul of Wash Buffer. Incubate for 2 min. Centrifuge at 15,000 xg for 30 sec.

10. Dry the column by centrifuging at  15,000 xg for 3 min.

11. Add 50 ul of Elution Buffer. Stand at 50°C in the Dry Bath  for 3 min.

12. Get the plasmid DNA by centrifuging at 15,000 xg for 2 min.