Prepared by Jessica Kerins from unknown original
*When working with EMS (or ENU), always work in fume hood and wear lab coat, goggles, and gloves.
1. When plates have at least 50% L4s, wash 6 plates with M9 buffer (1 mL/plate) and transfer with a glass Pasteur pipette to two 15 mL conical tubes.
2. Fill tubes with M9 buffer and spin in clinical centrifuge (setting 4, or 1800 rpm) for 4 minutes
3. Aspirate supernatant and fill each tube with 6 mL M9 buffer. Spin again.
4. Aspirate supernatant and fill each tube with 4 mL M9 buffer.
5. In fume hood, pour 20-25 mL of 4N NaOH in a 50 mL conical tube, for disposal of EMS-contaminated tips.
6. In fume hood, with a filtered tip, add volume (see table below) of EMS stock (stored at RT in fume hood) to the 15 mL conical tube of worms + M9. Shake thoroughly. Decontaminate tip by disposing in the tube of NaOH.
|Final EMS concentration||Volume of EMS stock added to tube|
We generally use a final EMS concentration of 25 or 50 mM and find that these concentrations give a reasonable mutation rate and little toxicity.
7. Incubate tubes for 4 hours at 20 C on a rotating shaker set at 100 rpm.
8. Spin tubes as above. Remove supernatant with Pasteur pipette, leaving 1 mL in the tube, and dispose both supernatant and pipette in NaOH.
9. Add 6 mL of M9 buffer to tubes and spin as above. Remove supernatant, leaving 0.5 mL in the tube, and decontaminate both supernatant and pipette as above.
10. Transfer remaining 0.5 mL onto a NGM plate (1 plate/tube). Let dry with lid ajar for 30 minutes in fume hood, or until all EMS has evaporated. Decontaminate conical with NaOH.
11. Transfer three L4s/plate onto 30 fresh plates and incubate at 20 C. Let self, then pick one L4/plate (as many plates as possible – we usually do about 3 big boxes of plates ~400-500 plates/box) and incubate at your desired screening temperature. Screen the next generation for an F2 screen.
12. Dispose NaOH/EMS solution down the fume hood sink. Dry waste can be disposed of normally once all EMS has evaporated.