WARNING: EMS is poweful mutagen and a suspected carcinogen. Wear gloves
and work in fume hood. Use disposable plasticware and inactivate mutagen
before disposal as outlined below.
- Grow worms (one to several plates) until most animals are in the L4
- Wash worms off plate with M9 into a plastic 15 ml conical
- Spin the worms in the clinical centrifuge on high for 30 sec. Aspirate
supernatant. The worm pellet may be very loose so be careful not to
- Wash once by adding M9 to the worm pellet, invert tube several times,
and repeat step 3.
- Resuspend worm pellet in 2 ml of M9.
- In the hood, make a solution of 0.1M EMS (ethyl methanesulfonate, also
known as methane sulfonic acid ethyl ester, Sigma #M-0880) by adding 20
l of liquid EMS to 2 ml of M9 buffer in
another 15 ml conical tube. Gently agitate until the dense oily liquid has
dissolved. Put the EMS pipet tip in a plastic 50 ml conical tube ("waste
- Add 2 ml of the suspension of washed worms to the 2 ml of EMS
solution (final concentration 0.05M EMS). Parafilm the top. Place the tube
on the rocker at 20°C for 4 hours. Keep the 15 ml and 50 ml conical
tube in the hood.
- After mutagenesis wash worms twice with M9 buffer. Put all pipet tips
and pipets in the 50 ml tube. Transfer worms, in a few drops of M9, using
pasteur pipette, to the edge of the bacterial lawn on an E. coli plate. Do
not ever transfer worms with a plastic tip -- they stick to the
- Inactivate EMS solutions by mixing them with equal volume of
"inactivating solution" (0.1M NaOH, 20% w/v
Na2S2O3 [sodium thiosulfate])
for 24 hours. All pipets/tubes contaminated with EMS should be soaked in
inactivating solution for 24 hours prior to disposal.
- Let the worms sit for 15-20 minutes (some wait 2 hours). Pick off the
healthy looking late L4 animals to use as P0's. Mutagenizing too early
(before much germline proliferation) will theoretically give less
independently mutagenized genomes, and give jackpots from those mutations
that do occur. Mutagenizing too late will be ineffective.
NOTE: Some people pick late L4's to mutagenize, so that the animals are
very young adults at the end of the EMS treatment. It is somewhat
difficult to recognize L4's after the EMS treatment; because the animals
are starved, the white crescent normally visible in the vulval region of
well fed L4's is not very apparent. The "dot" that appears within the
crescent at the very end of L4 is still visible in starved animals.