EMS Mutagenesis

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.

1. Grow worms (one to several plates) until most animals are in the L4 stage.

2. Wash worms off plate in M9 into a plastic 15ml conical tube.

3. 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 disturb it.

4. Wash once by adding M9 to the worm pellet, invert tube several times, and repeat step 3.

5. Resuspend worm pellet in 2mls of M9.

6. In the hood, make a solution of 0.1M EMS (ethyl metanesulfonate, also known as methane sulfonic acid ethyl ester, Sigma #M-0880) by adding 20 l of liquid EMS to 2ml of M9 buffer in another 15ml conical tube. Gently agitate until the dense oily liquid has dissolved. Put the EMS pipet tip in a plastic 50ml conical tube (waste tube).

7. Add 2mls of the suspension of washed worms to the 2ml of EMS solution (final concentration 0.05M EMS). Parafilm the top. Place the tube on the rocker at 20C for 4 hours. Keep the 15 ml and 50ml conical tube in the hood.

8. After mutagenesis wash worms twice with M9 buffer. Put all pipet tips and pipets in the 50ml  tube. Transfer the 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 inside.

9. 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.

10. 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.