Yeast DNA isolation with Lyticase or Zymolyase - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

This protocol does not include glass beads, so it should yield higher molecular weight DNA fragments. This protocol is based on Philippsen, P., Stotz, A., & Scherf, C. (1991). DNA of Saccharomyces cerevisiae. Methods in Enzymology, 194, 169–182.

1. Grow cells in 15 mL of YPD overnight at 30 ºC (final O.D. should be greater than 3)
2. Centrifuge cells at 5000 rpm for 5 min at 4 ºC. Discard supernatant.
3. Resuspend cells in 1 mL of sorbitol 0.9 M EDTA.Na2 (pH 7.5) 0.1M -> Transfer to eppendorf
4. Add 10 mL of lyticase (4 U/µL) and incubate for 1h at 37 ºC (or zymolyase 60 U/mL)
5. Centrifuge cells (spheroplasts/protoplasts) at 5000 rpm for 5 min. Discard supernatant.
6. Resuspend spheroplasts in 1 mL TrisHCl (pH 7.4) 50 mM, EDTA.Na2
7. Add 30 µL of SDS 10% and mix thoroughly.
8. Incubate at 65 ºC during 30 min.
9. Add 250 µL potassium acetate 5M and incubate on ice for 1h (or at -20 ºC)
10. Centrifuge at 10000 rpm for 10 min.
11. Transfer supernatant to 2 new tubes and add 1 volume (750mL) of cold isopropanol (or cold EtOH). Mix thoroughly by inverting the tube and centrifuge at 5000 rpm for 15 min.
12. Discard supernatant and incubate in 1 mL 70% ethanol for 5 min -> wash twice
13. Let pellet dry and resuspend in 200 µL of TE (pH 7.4) - this might take some hours, therefore, incubate 10 min at 42 ºC in order to help speed it up
14. Add 0.5 µL RNAse (1 mg/mL) and incubate for 30 min at 37 ºC
15. Centrifuge for 15 min at 10000 rpm. Transfer supernatant to new tubes.