Contributed by Ian Chin-Sang, Queens University, ON, Canada
EMS Mutagenesis protocol
EMS is a potent carcinogen. Use caution when dispensing. Double your gloves and if you get your gloves contaminated, remove by turning inside out and dispose of in the solid EMS waste. Any paper towel or kimwipes that are contaminated can be held in a gloved hand and the glove turned inside out to dispose of. All tips that come in contact with the EMS should be soaked in 1M NaOH solution prior to throwing away.
- Wash plates of worms (mixed staged or synchronized) with M9 buffer (1ml/plate).
- Put worms in a microfuge tubes. Spin for 1 min at high speed. Aspirate M9 and pool all worms into one microfuge tube. Resuspend (wash) in 1ml M9 and spin (use 1ml H2O as balance). Wash 2 more times.
- Resuspend worms in a volume of about 1ml M9. Add 5 micorliters of EMS (concentrated from bottle) to the 1ml of worms and mix immediately. This makes a final concentration of 50mM1 EMS (Note: 20 microliters of EMS in 4 ml M9 ~= 47mM).
50mM EMS is standard, but others have found this to be too high. Vary concentrations from 10 to 50mM EMS. If large volumes of worms are going to be used then carry out the EMS mutagenesis in 4 ml of M9 and use a 15ml polypropylene tube.
- Incubate worms at 22C for 4-5hours with mild rocking. Use the “labquake” rocker
- Spin 30 seconds. Aspirate off supernatant making sure not to suck up the worms. Note, you must use the vacuum with the side arm flask trap. Put a solution of 2M NaOH in the trap so the EMS will be inactivated. Wash worms at least 4 times with 1ml M9 buffer.
- Resuspend worms in an appropriate volume of M9 and use a glass pipette to plate out onto seeded plates. Let recover 4 hours to overnight and pick young adults (3-5) to separate plates.
- From these plates pick individual F1s to separate plates. Score for F2 mutants 4 to 5 days later.
This method is based, with permission, on an original protocol available here.