EMS Mutagenesis

For mutagenesis, one wants to prepare 6-10 plates of animals enriched for the L4 stage. Pick plates of 4 or 5 L4 animals 4 days before mutagenesis and put in 20 C incubator. In the end you only need about 4 plates for mutagenesis.

1. Prepare a solution of 0.1 M NaOH, about 500 ml in a plastic beaker. This will be used to neutralize the EMS from pipette tubes, tubes. LABEL THIS CONTAINER with your name, the date and “SODIUM HYDROXIDE”.
2. Wash each plate with 1 ml M9. Spin down gently in picofuge.
3. Collect worms in 15 ml conical tube using glass pippetes (they are in the hood in metal boxes, prevents worms from sticking to tube). Have a final volume of 2 ml in this tube.
4. Add 2 ml of M9 into another 15 ml conical tube. Add 20 ul of EMS into this solution.
5. Vortex EMS-M9 tube until EMS dissolves.
6. Pour the 2 ml solution of EMS-M9 into conical tube with worms.
7. Put on rotator for 4 hrs.
8. Add 10 ml of M9 to tube. Spin down gently using centrifuge in hood (setting 1 or 2 for 10 secs.). Aspirate supernatant with glass pippetes. Repeat 5 times.
9. Suck up worms using a glass pipet and plate onto seeded plates. Let the worms rest for 2 hrs.
10. Pick mid to late L4s onto a new plate. These are your P0s.
11. Age the animals 24 hrs after the end of EMS treatment. This will allow you to avoid the first eggs which may have already been meiosis at the time of mutagenesis.
12. Pick P0s onto a new plate.
13. Pick L4 F1s.
14. Score F2s.
15. Dispose of pipette tips, tubes, in normal trash 24 hrs after EMS mutagenesis. Dump 0.1 M NaOH down the sink.