Plasmid Prep from Yeast

1. Inoculate yeast overnight culture in ~ 4 ml selective media (e.g., SC-Leu or –Ura w/ 2%D)      OPTIONAL (may help); dilute the overnight in the morning to OD 0.2 and let grow until in log phase (OD ~0.8 - 1.0)

2. Spin 1.5 ml for 10 min at 5000 rpm in microfuge tube, and remove supernatant (supe)

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

4. Resuspend pellet in 100ul of 5mg/ml Zymolase in ~300mM Tris pH 7.5 (make fresh)

5. Let incubate 30 min at 30C

6. spin 5min 5000 RPM, remove supe

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

8. Add 250 ul Qiagen buffer P2

9. Add scoop of acid washed glass beads

10. Vortex for 1 min.

11. Let sit 5 min in P2 on ice.

12. Add 350 ul chilled Qiagen buffer N3

13. Mix by inverting 4-6 times, then incubate on ice for 5 min.

14. Spin 10 min, full speed

15. Place Qiaprep - spin column in 2 ml microfuge tube and add supe to column (after the spin)

16. Spin 30-60 sec, discard flow through

17. Wash column with 500µl Qiagen buffer PB

18. Spin 30-60 sec, discard flow through

19. Wash column with 750 µl Qiagen buffer PE

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

21. Place column in clean 1.5 ml microfuge tube

22. Elute by adding 50ul EB to center of column, let sit 1-2 min, spin 30 sec

23.  Nanodrop to determine concentration and check peak to make sure DNA is clean

24a. (Typically) - Transform isolated plasmid DNA into E.coli using standard E.coli transformation. 24b. (Possible) - If yields are good (e.g. > 40ng/µl and peaks are clean, might try directly sequencing yeast plasmid prepped DNA)