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To screen mutant populations for altered eclosion rhythms, fly stocks were maintained in vials at 18°C under a LD 12 12 schedule for at least 5 days prior to adult eclosion.
Pure cultures were maintained in vials with 20% glycerol at −80˚C.
Flies were maintained in vials at a density of ten flies per vial.
For stocks maintained in vials, 25 females were kept for stocks with 10 males at maximum to prevent over-crowding.
This population, like all others used in this assay, is maintained in vials on a discrete 14-day culture cycle.
All Drosophila stocks were maintained in vials containing Fortified (F1) medium and kept at either 18 or 25°C.
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Briefly, samples of 2.5 g culture were placed in vials, they were warmed for 15 min at 65 °C, and the volatiles were extracted at a pressure at 207 kPa maintained in vial for 1 min with the carrier gas (helium), before being adsorbed on a Tenax™ trap at 35 °C.
For controlled crosses, virgin female flies were collected and maintained in polypropylene vials containing fresh CMYA medium, with no more than 20 copies per vial.
A single fly was maintained in a vial and each vial was tipped on every 1 3 days.
These sets of three individuals were maintained in single vials at 25 °C, under 12 h of light and 12 h of dark cycles and food vials were changed every week until they have reached the age required to perform the experiments.
The pair was maintained in plastic vials as described above (4 + 2 + 2 + 2 days).
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