Procedures:

Note: EMS is a potent mutagen! Use proper clothing, gloves, containment tray, etc. Sodium thiosulfate should be used to neutralize any EMS spills and to neutralize treatment solutions after mutagenesis. All mutagenesis procedures should be carried out in a fume hood.

1. Weigh out spores to be mutagenized and transfer to a 15 mL conical centrifuge tube.

2. Mix appropriate amounts of buffer and EMS, and vortex very thoroughly and carefully. NOTE: It is very important to thoroughly vortex the mixture because EMS is difficult to dissolve in buffer. If EMS solution is properly mixed, spores should mostly sink to the bottom after wetting and settling. If spores are mostly floating, mixing was not adequate.

3. Treat spores for 36 hours. Remove EMS solution and wash spores three times with 2 mL distilled H2O using the technique described in the SPORE STERILIZATION section.