Yeast plasmid purification

A method for plasmid purification directly from yeast.

Singh and Weil. Analytical Biochemistry 307, 13-17.

Materials

• Lyticase from ICN Biomedicals
  • 1.2 M sorbitol
  • 0.1 M NaPO4 buffer, pH 7.4
  • 5 mg/ml Arthrobactor luteus lyticase. (can substitute an equal mass of Zymolyase)
• Qiagen Miniprep Kit

For 1 L yeast grown overnight in YPD media

1. Pellet at 2000 x g for 5 minutes at 4 °C
2. Half the pellet (?) in 5 ml P1 Buffer
3. Add 5 ml Lyticase (1.2 M sorbitol, 0.1 M Na PO4 Buffer pH 7.4, 5 mg/ml Arthrobactor luteus lyticase)
4. Incubate at 37 °C for 45 minutes
5. Add 10 ml P2, mix gently by inverting
6. Incubate at RT for 10 minutes (becomes viscous)
7. Add 14 ml N3, mix gently by inverting
8. Incubate on ice for 30 minutes
9. Centrifuge at 10,000 x g for 10 minutes at 4 °C
10. Apply supernatant to “several” miniprep columns
11. Wash once with 500 µl PB
12. Wash once with 750 µl PE
13. Spin once to remove residual ethanol
14. Add 50 µl elution buffer, incubate 5 minutes at RT
15. Spin 1 minute
16. Add an additional 35 µl elution buffer, incubate 5 minutes at RT
17. Spin 1 minute

For small cultures (5-10 ml YPD or 30-40 ml 20 °C SDCAA o/n).

1. Resuspend in 200 µl P1
2. Add 100 µl Lyticase
3. Incubate at 37 °C for 20-30 minutes
4. Add 300 µl P2, mix gently by inverting
5. Incubate at RT for 10 minutes
6. Add 420 µl N3, mix gently by inverting
7. Apply supernatant to a miniprep column
8. Wash once with 500 µl PB
9. Wash once with 750 µl PE
10. Spin once to remove residual ethanol
11. Add 50 µl elution buffer, incubate 5 minutes at RT
12. Spin 1 minute
13. Add an additional 35 µl elution buffer, incubate 5 minutes at RT
14. Spin 1 minute