Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 298

E. Isolation of Embryos and Synchronization as Starved L1 Larvae
The washed worms are bleached to isolate embryos ( Fig. 4 ). Good bleaching
efficiency depends on small volumes (maximum 5 mL worm pellet per 50 mL
conical) and freshness of bleach. Bleaching should be followed by eye under a
dissecting microscope. Bleaching sterilizes the culture; after bleaching, sterile tech-
nique becomes critical again so that no contaminants are introduced into the material
that will be used to inoculate the synchronized liquid culture.
1. To worm pellet add 25 mL 0.1 M NaCl and mix by pipeting up and down twice.
Settle worms on ice for 5 min.
2. Aspirate off supernatant including worms that have not settled to the bottom and
add 0.1 M NaCl up to a volume of 30 mL.
3. Mix 5 mL 5 N NaOH with 10 mL bleach in conical. Immediately add NaOH/
bleach mix to 30 mL worm suspension.
4. Vortex at maximum speed for 5 s and stand tube at RT for 2 min. Repeat four
times for a total bleaching time of 7-9 min. Follow bleaching by examining
samples on a glass slide under a dissecting microscope. Stop bleaching when
only embryos remain. The color of the bleach mixture will change to a burnt
orange as worms are dissolved.
5. Immediately centrifuge at 800g for 1 min at 4 C. Aspirate off supernatant.
6. Add sterile water to a total volume of 50 mL, mix by inverting the tube, and
centrifuge at 800g for 2 min.
7. Repeat step 6. Washed embryos from synchronized liquid culture can be frozen
for immunoprecipitation as described in Section III.H.
8. Add 35 mL M9 and transfer to a 50 mL flask. Rinse the conical with an extra
10 mL of sterile M9 and add to flask. Shake at 20 C until embryos hatch and are
starved L1 larvae ( 18-20 h).
F. Seeding Synchronized Cultures Using Starved L1 Larvae
Starved L1 larvae from 500 mL starter flask can be expanded to inoculate up to six
synchronized liquid cultures.
1. 2 Days before: Start 300 mL culture of OP-50-1 in LB + 50 m g/mL streptomycin.
2. Day before: Start six 1.5 L cultures of OP-50-1 in LB + 50 m g/mL streptomycin.
3. Harvest bacterial cultures at 4200 rpm for 15 min in sterile 1 L centrifuge
bottles. Pour off LB.
4. Make 3 L Complete S Basal and distribute into six 2.8 L Fernbach flasks.
5. Resuspend bacterial pellet of each 1.5 L culture in 20 mL Complete S Basal and
transfer into sterile 2.8 L Fernbach flask with 500 mL Complete S Basal.
6. When flasks with bacterial food are ready, start processing starved L1s. In the
hood, transfer L1s to a 50 mL conical. Chill on ice for 5-10 min.
7. Spin at 600g for 3 min. Carefully remove supernatant. Bring up to 50 mL with
sterile cold M9.