1. Clear a vial of any adult files by inverting the vial over a fresh vial or a vessel containing ethanol and tap the adult files into the new vessel.

2. Replug the vial and put it back into an incubator or culture room.

3. At 25 C virgins need to be collected 10-12 hours after clearing of adult flies. At 18 C females will stay virgins for about 16 hours. Collect vigins and keep them in a fresh vial. Don't keep too many virgins in one vial since all files will be discarded if there is contamination of one male.

4. Females lay eggs ~48 hours after eclosion. Let them sit in a vial for 3-4 days. If any of the flies is not virgin, first instar larvae will be visible.

5. Virgins older than 10 days are commonly not used for mating.