S. pombe Genomic DNA Midiprep (zymolyase method)

1. Materials

Zymolyase: Chris Yellman in the Marcotte lab said: The enzyme is specified by activity level, and that is a 100T activity enzyme. You may find zymo from different suppliers and I think it will work just as well, but pay attention to the activity level number so that you know how much to use. I think the enzyme is also frequently sold with activity level 20T. In the past we used zymolyase from Seikagaku but they discontinued its production.
Yeast lysis solution: 1X TE, 2% SDS, 1M NaCl. You will need to microwave for 10-30 seconds until dissolved.
SCE
PCR cleanup kit

Grow and harvest the cells
1. Grow cells overnight in liquid culture to stationary phase (OD₅₉₅ of 1.5 is probably preferable, but 2.5 worked once).
2. Add 1ml cell culture to a microcentrifuge tube. Centrifuge for 1 min at 5000 RPM.
3. Remove the supernatant from each tube, add 1ml cell culture again, and repeat the centrifugation.
4. Discard supernatant from each tube.
Digest the cell walls with zymolyase
1. Resuspend each sample in 96µl SCE and 4µl of 10mg/ml (100T activity) zymolyase. Vortex to mix.
2. Incubate at 37°C with occasional gentle shaking.
3. Digestion should occur after 30-90 minutes. Cells should be dark grey under a phase contrast microscope.
Lyse the spheroplasts and precipitate protein
1. For each sample, prepare 100µl yeast lysis solution and add to each tube. Vortex for a few seconds.
Purify the DNA using a standard PCR cleanup kit
1. Add 500µl of PB solution (from PCR cleanup kit) to each sample.
2. Spin at maximum speed in microcentrifuge for 5 minutes to pellet the cell debris and precipitated protein.
3. Apply the clear supernatant to a DNA minicolumn and proceed with purification according to the kit’s instructions.
Store DNA
1. Leave the DNA in the elution buffer and store at -20°C.
Diluting the DNA
1. The resulting product can be diluted at least 100x and still be viable for PCR. Further dilution is probably possible.