Experimental crosses maintained at 25 o C should be discarded after 18 days to prevent recovery of second generation progeny. Some mutant phenotypes are affected by temperature or genetic background. Before setting up a large scale effort such as a screen, make a test cross of the relevant genotypes under the conditions to be used and confirm that all phenotypes are scorable. It is also prudent to assure the absence of 'background' lethals in a stock to be used for mutagenesis by isogenizing a chromosome for use in a screen. Only lines carrying lethal-free chromosomes will produce homozygotes among the progeny of the sib matings.
Most experimental schemes require virgin females.
D. melanogaster adults do not mate for about 10 hours after eclosion, allowing virgins to be collected within 8-12 hours after the culture has been cleared of adults. The timing of this virgin window appears to be genotype dependent. Collect females within 8 hours to be safe, or determine empirically for a given strain how long you can wait and still recover virgins. For many mating schemes virginity is desirable for efficiency's sake, but not essential because the progeny of non-virgin females can be distinguished phenotypically from the progeny of interest. The peak of female fertility is genotype-dependent, but on average females are best used between 4 and 10 days old.