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EMS mutagenesis

EMS mutagenesis

by Michael Koelle


1. Wash worms off plates with M9, using sterile glass pipettes. Want to use plates with a lot of early L4 larvae. Transfer to a 15 ml sterile plastic centrifuge tube. Spin down (1000 rpm in a clinical centrifuge for 30 sec) & remove sup. or add M9 until you have a total of 3 ml of worm suspension in the 15 ml sterile plastic centrifuge tube. Most people in the Horvitz lab wash the worms once or twice in M9 (by spinning down, removing the supernatant, and resuspending) to remove the bacteria; other people don't bother and they get mutants just fine.

2. Place 1 ml M9 in a separate 15 ml tube. Wearing gloves, in a hood, using a special pipette used only for EMS, add 20 µl EMS (methanesulfonic acid, ethyl ester, Sigma #M-0880). EMS is stored at room temp. in the hood. Keep a special jar for sharps (pasteur pipettes and pipettman tips) and another one for gloves that have been exposed to EMS. Shake the EMS suspension until it is no longer cloudy.

3. Transfer the 3 ml of worms into the tube of EMS. Parafilm the top. Place the tube on a spinning wheel at 20° for 4 hours. The final concentration of EMS is 47 mM.

4. Spin the worms down (keep a special grungy old clinical centrifuge for EMS work only) and remove the supernatant to a 15 ml plastic centrifuge tube. Wash the worms twice with ~3 ml M9, transferring the supernatants to the other 15 ml tube. Transfer the worms, in a few drops of M9, using a sterile glass pipette, to the edge of the bacterial lawn on an E. coli plate. Do not ever transfer worms with a plastic yellow tip- they stick to the inside. Kill the EMS in the the liquid from the washes by adding a few pellets of KOH. Mix a little, label the tube, and leave in the hood for a day. Then pour down the sink and discard the tube in the trash.

5. 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. 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.

M9 medium (w/o glucose or MgSO4)

5.8 g Na2HPO4

3.0 g KH2PO4

0.5 g NaCl

1.0 g NH4Cl

Bring to 1 liter w/ dH20. Dispense into 100 ml bottles and autoclave.