Skip to main content

A Dohlman Lab Protocol

1. Grow culture to saturation in 4 ml selective media

2. Spin 1.5 ml for 10 min in microfuge tube, and remove supe

3. Add another 1.5ml of culture to pellet, spin, and remove supe (optional)

4. Resuspend in 500 ul sterile dd H2O

5. Spin 10 min, remove supe

6. Resuspend pellet in 250 ul Qiagen buffer P1 (containing RNAse A)

7. Add 250 ul Qiagen buffer P2 and 250 ul acid washed glass beads

8. Vortex for 2 min. Let sit 5 min in P2. Do this at 4C.

9. Add 350 ul chilled Qiagen buffer N3

10. Mix by inverting, then incubate on ice for 5 min.

11. Spin 10 min

12. Place Qiaprep – spin column in 2 ml microfuge tube and apply supe (after the spin)

13. Spin 30-60 sec, drain tube

14. Wash column with 0.5 ml Qiagen buffer PB

15. Spin 30-60 sec, drain tube

16. Wash column with 0.75 ml Qiagen buffer PE

17. Spin 30-60 sec, drain tube, spin again

18. Place column in clean 1.5 ml microfuge tube

19. Elute DNA with 50-100 ul TE (10 mM Tris-HCl pH 8.5, 1 mM EDTA) or dd H2O

20. Spin 30 sec

21. Transform a range of plasmid amounts into E. coli to amplify

ALTERNATIVE METHOD: We have started using the Zymoprep Yeast Plasmid Miniprep Kit II with much success.  Make sure you select Miniprep II as the kit.