From Wikionchus

ATTENTION: The procedures described in this section invovle handling dangerous substances: EMS can do to you what it should do to the worms. Do your first experiments together with an experienced fellow. Work under the hood and inform yourself about protective measures.

Routinely used mutagens

• EMS
• ENU
• TMP-UV

F2 EMS screen for egg-laying defective mutants

• you need 4-5 full plates with mainly J4 animals

• wash them with M9 into a 15 ml falcon tube, let them settle(or gently centrifuge) and replace M9 to wash. Repeat this step twice.

• resuspend in 2 ml M9

• in another falcon tube, pipette 2 ml M9 and 20 ul EMS and mix well

• fuse the contents of the two tubes: you have 20ul EMS in 4ml M9, obtaining a 50mM EMS solution

• wrap tube in parafilm, put it in double-layered plastic bags and incubate 4 hours at 20°C in horizontal position

• Inactivate supernatant in NaOH. Wash worms 3 times in 6ml M9, always inactivate supernatants in NaOH.

• Resuspend worms in 0,5 ml M9 and spread on 1-2 6cm NGM plates without OP50

• When plates are dry (after 1-2 hours) put single healthy J4 animals on 10 cm NGM plates spotted with 2 ml OP50. (1worm / plate)

• Kill P0-animal after it laid 20-30 eggs. At 20°C this takes at least 12 hours, check frequently!

• When the second generation is aging (roughly 8-11 days after picking the P0) pick egg-laying defective animals.

Links

• Back to Pristionchus pacificus Protocols page