Plasmid Miniprep with Spin Column
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.