Purification of Plasmid from 50 ml-culture

 

1. Shake E. coli harboring plasmid at 37 C overnight in 50 ml of TB containing appropriate antibiotics. (when using ampicillin, addition of the antibiotics to 100-200 ug/ml rather than usual 50 ug/ml may improve the yield of plasmids.)

2. Spin the bacterial culture at 6,000 rpm for 10 min at 4 C. Discard the supernatant.

3. (0ptional) Suspend the cell in about 20 ml of deionized water. Spin again. Discard the supernatant. Remove all of the supernatant fluid using pipetman.

4. Resuspend the pellet in 5 ml of Solution I.

5. Add 10 ml of Solution II. Mix well by inverting the bottle more than 10 times. The solution should be clear after mixing Solution II.

6. Add 7.5 ml of Solution III. Mix well as above.

7. Spin the lysate at 10,000 rpm for 10 min at 4 C.

8. Transfer the supernatant into a new bottle. Add about 15 ml of isopropanol, mix well, and store the bottle for 10 min at room temperature.

9. Spin at 10,000 rpm for 10 min at 4 C. Discard the supernatant. Remove all of the fluid using pipetman.

10. Dissolve the pellet in 600 ul of TE. Transfer the solution into a microfuge tube.

11. Add 200 ul of 8M LiCl. Mix well, and then spin the solution at 14,000 rpm for 5 min at 4 C.

12. Transfer the supernatant containing plasmid DNA to a new microfuge tube. Add 600 ul of isopropanol. Mix well, and then spin the solution at 14,000 rpm for 5 min at 4 C.

13. Discard the supernatant. Rince the pellet and the wall of the tube with 500 ul of cold 70% ethanol. Discard the fluid.

14. Add to the pellet 400 ul of TE containing DNase-free RNase A (20 ug/ml). Incubate the tube for 30 min at 37 C.

15. After 30 min, carefully check the content of the tube. If nucleic acid pellet is visible at the bottom of the tube, vortex well to dissolve the pellet. Incubate the tube at 37 C for further 30 min.

16. Add 240 ul of 2M NaCl, 20% PEG8000. (PEG6000 supplied from Japanese suppliers is essentially equivalent to PEG8000, and works well.)

17. Spin at 14,000 rpm for 5 min. Discard the supernatant. Rinse the pellet with 300 ul of cold 70% ethanol. Discar the fluid. Dissolve the pellet in 400 ul of TE.
(Optional: Repeat PEG precipitation once more. This is recommended for preparing dephosphorylated linear vector since trace amount of short RNA in the vector preparation may interfere with the dephosphorylation reaction.)

18. Extract the plasmid solution with chloroform to remove PEG. Take aquaous phase. Extract the aquaous phase with phenol. Take aquaous phase.
(Optional: Repeat phenol extraction until no interphase is visible. This is recommended for preparing the template for in vitro transcription.)
Extract the aquaous phase with chloroform or ethylether to remove trace amount of phenol dissolved in the solution. Take aquaous phase (or remove organic phase).

19. Add 0.1 volume of 3 M sodium acetate and 3 volume of ethanol into the plasmid solution. Spin at 14,000 rpm for 5 min at 4 C. Discard the supernatant. Rinse the pellet with 200 - 500 ul of 70% ethanol.

20. Store the open tube on the bench until the visible traces of ethanol have evaporated.

21. Dissolve the DNA pellet in 200-500 ul of TE. Typically 300-800 ug of plasmid should be obtained if the plasmid have pUC-based replication origin.

SOLUTIONS

Solution I

50 mM glucose
25 mM Tris-Cl (pH 8.0)
10 mM EDTA (pH 8.0)
Autoclave, and store at room temperature.

Solution II

0.2N NaOH
1% SDS
Store at room temperature in a plastic bottle. Don''t autoclave.

Solution III

5M potassium acetate 60 ml
glacial acetic acid 11.5 ml
Distilled water 28.5 ml
Store at room temperature. This solution can be autoclaved. However, we usually use this solution without autclaving.