Mutagenesis of C. elegans

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Materials:

ENG plates	0.5% Peptone 0.1% Yeast Extract 51 mM NaCl 2% agar add after autoclaving: 5 ug cholesterol, 1 mM CaCl2, 1 mM MgSO4, 1 mM K-phosphate (pH 6)
1 M NaOH	make up 500 mls in plastic container
1 M NaOH waste container
M9 Buffer	22 mM KH2PO4 42 mM Na2HPO4 85.5 mM NaCl 1 mM MgSO4
EMS (Ethyl Methanesulfonate)	(Sigma M-0880)

Procedure:

1. Grow up P0 generation to L4 stage
   • chunk starved plates of worms onto a few 100 mm ENG plates and grow to gravid adults
   • obtain synchronous population of worms by bleaching gravid adults and hatching off embryos
   • innoculate 1-10 100 mm ENG plates with synchronized population of L1s (=P0 generation) at a density of 5,000 - 10,000 per plate
   • grow to L4 stage (around 36-42 hours @ 25 degrees)
2. Mutagenize P0 generation
   • harvest L4s from plates with M9 and spin down (20-30 seconds at 150-200 x g) in 15 ml conical tube(s)
   • wash once with M9 to remove bacteria
   • resuspend pellet of worms in 2 mls M9
   • prepare 2X EMS solution by adding 12 ul EMS to 2 mls M9 solution (=60 uM EMS) and mix well to get EMS into suspension
     • decontaminate pipette tip by rinsing in 1 M NaOH
   • add 2 mls 2X EMS solution to 2 mls worm suspension (gives final 30 uM EMS concentration)
     • decontaminate pipette by rinsing in 1 M NaOH
   • tightly cap conical tube and wrap with parafilm to ensure seal
   • place worms in EMS solution on rocker in hood for 4 hours
   • remove EMS solution by spinning down worms 30 seconds (hood centrifuge setting between 1/2 and 3/4)
   • remove supernatant and discard into 1 M NaOH waste bucket
   • wash worms 2X with 4 mls M9
   • resuspend worms in 0.5 mls M9 and plate onto seeded 100 mM ENG plates
   • leave in hood with lids slightly open to allow evaporation of any remaining EMS
   • grow mutagenized worms to gravid adults (typically O/N after mutagenesis)
3. F1 screen:
   • harvest gravid P0 adults
   • bleach to obtain F1 embryos and hatch off to synchronize
   • plate out F1s onto seeded 60 mm NG plates at a density of approx 100-200 worms per plate
   • place under non-permissive conditions or other selection
4. F2 screen:
   • harvest gravid P0 adults
   • bleach to obtain F1 embryos and hatch off to synchronize population
   • plate out F1s onto seeded ENG plates at a density of 10,000 per plate
   • grow to gravid adults and harvest
   • bleach to obtain F2 embryos and hatch off to synchronize population
   • plate F2 worms onto seeded NG plates - only use 5% of population to avoid duplicates
   • place under selective conditions to score desired phenotype

Notes & Misc:

• Decontaminate everything that has come in contact with EMS by rinsing with 1 M NaOH before placing in biohazardous waste - rinse pipettes, gloves, tubes, etc.
• Use freshly seeded ENG plates (1-2 days old) to avoid too thick OP50 lawns
• Can also use final 50 uM EMS for stronger mutagenesis but increases "background" hits
• To facillitate embryonic lethal screens, perform in lin-2 background and pick gravid F2 worms that are do not show the "bag-of-worm" phenotype
• him-3 backgrounds are useful for a low fraction of males when it comes time for outcrossing. him-3 gives roughly 3% males - low enough not to compromise mutagenesis but high enough to obtain a few males

compiled by Chad Rappleye

Aroian Lab Protocols