Editing Plasmid Miniprep with Vacuum

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

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.<br> 

4. Label Falcon tubes by number with red marker pen.<br> 

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. <br> 

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. <br> 

10. Spin at 13000 rpm&nbsp; for 2.5 min, two times. <br> 

11. Label spin columns by number with red marker pen.<br> 

12. Transfer clarified liquid into a spin column<br> 

13. Put spin columns onto a QIAvac 24 Plus, turn on pump.<br> 

14. Wash spin column with '''350''' µl PB.<br> 

15. Wash spin column with '''350''' µl PE. '''Repeat''' once. <br> 

16. Put spin column onto a collection tube, spin at max speed for 1 min to remove ethanol.<br> 

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

18. Add '''50''' µl autoclaved ddH<sub>2</sub>O or EB (for low copy number vector such as pET28, pET30) or '''75''' µl ddH<sub>2</sub>O or EB (for high copy number vector such as lentivirus, adenovirus and AAV vector).<br> 

19. Spin at max speed for 1 min. 

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

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

22. Store plasmids in a freezer box at -20 <sup>o</sup>C. 

<br>

use ultiscreen glassfilter FB,1.0μm pore, Millipore, USA for high througput miniprep<br>

[[Category:Protocols]]