MiniPrep using Spin Column

Buffer

P1, P2, N3, PG, PE can be found on Plasmid_buffers page

1. Grow Ecoli from glycerol stock in 5 ml or 15 ml LB or TB medium with antibiotics

or pick a single colonies from plate transformation, inoculate in liquid medium

2. Grow  E coli  at 37 oC o/n in a shaker-incubator, 180 rpm

3. Label each tube using red marker pen. Spin down 3000 rpm 3~5 min

4, dump off liquid

5. Add 250 ul P1, resuspend by votexing, make sure it is a homogeneouss liquid suspension 

6, Add 250 ul P2. Mix gentely until completely lysed, do not votex

7. Add 350 ul N3, mix by shaking or votexing

8. Transfer lysates to 1.5 ml tubes by pouring., numbered with red marker pen

9, Spin at 3~5 min at max speed, 2 times, turn microcentrifuge tube in opposite directoion each time

10. Transfer to spin column by pouring

11. Spin 30 sec at max speed, dump off liquid

12. Wash column with 350 ul PB, dump off liquid

13, Wash column with 2X 350 ul or 700 ul PE, dump off liquid

14. Spin another min at full speed, discard collection tube, transfer spin columns to new autoclaved tubes numbered with red marker pen

15. Add 55 ul TE or EB, do not cross contaminate

16. Spin at full speed for 1 min.

17. Check DNA quality and conc using nanodrop

18. Label each tube using printed label

19. Store at -20 oC.