Biology Reference
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8. Repeat step 7.
9. Add sterile M9 such that total volume is 5 mL, transfer L1s to 15 mL conical,
and pellet by spinning at 600g for 2 min. Immediately after the spin, use a pen to
mark the volume of the pellet on the side of the tube. Estimate the volume of
pellet by adding known volumes of water to a separate conical.
10. Resuspend L1s to total volume of 12 mL using sterile M9 and look at a sample
under dissection microscope; estimate percentage of L1s relative to dead
embryos/worm parts/clumps.
11. Seed each flask with equivalent of 50 m L pure L1 pellet (e.g., if pellet volume is
0.6 mL and % L1 in the resuspension is 70% then seed with 1.4 mL of the
resuspended pellet). Avoid overseeding or cultures will starve.
12. Put flasks at 20 C at 230 rpm. Grow for 48 h while monitoring cultures under
dissecting microscope.
G. Freezing Adult Worms for Immunoprecipitation
Once the liquid cultures are ready collect and wash worms as described in Section
III.D. Wash worms by adding 50 mL cold 1 Lysis Buffer (with protease inhibitors).
Remove Lysis Buffer until only a small amount remains. Freeze adult worms by
dispensing from a pipette drop by drop in liquid nitrogen, which will form small
beads ( Fig. 4 ). Store at 80 C.
H. Freezing Embryos for Immunoprecipitation
Bleach adult worms as described in Section III.E. and continue after step 7. Wash
embryo pellet with 50 mL cold 1 Lysis Buffer (with protease inhibitors). Freeze
embryos by dispensing from a pipette drop by drop in liquid nitrogen. Store embryo
beads at 80 C.
I. Enriching for Specific Age Embryos
While precise synchronization of embryos is not possible, it is straightforward to
enrich for old or young embryo populations by varying growth conditions and
carefully monitoring worms in the culture under a dissecting microscope. Generate
synchronized starved L1 larvae (see Section III.E.) and inoculate worm cultures (see
Section III.F.). To obtain worms that just started embryo production, incubate the flask
for 64 h at 17 C. Using these growth conditions worms typically contained up to
five embryos, the majority of which have < 50 cells ( Fig. 5A ). If several flasks of
worm culture are grown simultaneously, it is necessary to monitor each flask sepa-
rately, as the time at which embryo production begins may vary between flasks. It is
also critical to avoid contamination, which may adversely affect synchronous growth
of the worms. To obtain worms with mostly old embryos ( > 200 cells), cultures are
incubated for 64 h at 19 C( Fig. 5A ). Embryos are frozen as described in Section
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