EMS Mutagenesis

 

<center> <p> <font>EMS Mutagenesis</font></p> </center>

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 several plates of worms 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 clinical centrifuge up to 1700 rpm. Do not use brake. Aspirate supernatant. The worm pellet may be very loose so be careful not to disturb it.

4. Add M9 to the worm pellet, invert tube several times, and repeat step 3.

Resuspend worm pellet in 2mls of M9.

5. In the hood, make a solution of 0.1M EMS (ethyl metanesulfonate, also known as methane sulfonic acid ethyl ester) by adding 20 ul of liquid EMS to 2ml of M9 buffer in a plastic 50ml conical tube. Gently agitate until the dense oily liquid has dissolved.

6. Add 2mls of the suspension of washed worms to the 2ml of EMS solution (final concentration 0.05M EMS). Incubate 4 hours at 20oC. Agitate occasionally to increase aeration. After mutagenesis wash worms four times with M9 buffer. Distribute worms to NGM plates.

7. Inactivate EMS solutions by mixing them with equal volume of "inactivating solution" [0.1M NaOH, 20% w/v Na2S2O3 (Sodium Thiosulfate)] for 24 hours. Pipets/tubes contaminated with EMS should be soaked in inactivating solution for 24 hours prior to disposal.