Zymoprep protocol - glasgowlab/home GitHub Wiki

A method for plasmid purification directly from yeast.
Singh and Weil. Analytical Biochemistry 307, 13-17.

Materials

  • 5 mg/ml Arthrobacter luteus lyticase stock solution in 1.2 M sorbitol, 0.1 M NaPO4 buffer, pH 7.4. (Equal mass of Zymolyase also OK. Stored in -20C.)
  • miniprep buffers.

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
  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. Centrifuge at 10,000 x g for 10 minutes
  8. Apply supernatant to a miniprep column
  9. Wash once with 500 µl PB
  10. Wash once with 750 µl PE
  11. Spin once to remove residual ethanol
  12. Remove bottom part of miniprep column and placed miniprep filter on 1.5 ml tube.
  13. Add 50 µl elution buffer, incubate 5 minutes at RT
  14. Spin 1 minute
  15. Add an additional 35 µl elution buffer, incubate 5 minutes at RT
  16. Spin 1 minute