Paul.xu at 20:31, 15 September 2011

2011-09-15T20:31:02Z

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1. Inoculate '''4''' mL LB/Amp or Kan in 15 ml Falcon tube with bacteria from a single colony 

2. Grow at 37 ºC overnight.

3. Take '''50''' µl cells out from each tube, put the liquid culture into a clean autoclaved 1.5 ml tube. 

4. Label Falcon tubes by number with red marker pen. 

5. Remove caps, spin cells at 3000 rpm for 5 min at RT.

6. Resuspend cells in '''250''' µl '''P1''' by votexing.

7. Add '''250''' µl '''P2''', shake gently until homogenous and clear. 

8. Add '''350''' µl '''N3''', mix by shaking.

9. Transfer liquid to a new 1.5 ml tube ('''pre-label the tubes by number with red marker pen'''), put the capped tubes on dry ice for 1 min. 

10. Spin at 13000 rpm  for 2.5 min, two times. 

11. Label spin columns by number with red marker pen. 

12. Transfer clarified liquid into a spin column 

13. Spin for 15 seconds, remove liquid 

14. Wash spin column with '''350''' µl PB, spin for 15 sec,  remove liquid 

15. Wash spin column with '''350''' µl PE, spin for 15 sec, remove liquid, '''repeat '''once. 

16. Spin at max speed for 1 min to remove ethanol. 

17. Transfer spin column into numbered 1.5 ml tube. Put the collection tube into a container (do not discard it). 

18. Add '''50''' µl autoclaved ddH2O or EB (for low copy number vector such as pET28, pET30) or '''75''' µl ddH2O or EB (for high copy number vector such as lentivirus, adenovirus and AAV vector). 

19. Spin at max speed for 1 min.

20. Label plasmid tubes by clone number, colony number and name, print labels if required. 

21. Verify plasmid by restriction digestion, PCR or sequencing.

22. Store plasmids in a freezer box at -20 oC.

[[Category:Protocols]]

Plasmid Miniprep with Spin Column - Revision history

Paul.xu at 20:31, 15 September 2011