Plasmid Miniprep with Vacuum
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. Put spin columns onto a QIAvac 24 Plus, turn on pump.
14. Wash spin column with 350 µl PB.
15. Wash spin column with 350 µl PE. Repeat once.
16. Put spin column onto a collection tube, 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.
use ultiscreen glassfilter FB,1.0μm pore, Millipore, USA for high througput miniprep