step	problem	possible reason	solution
14	Many eggs do not hatch	1. Did not allow enough time to hatch 2. Overbleaching 3. Not enough M9 was added or tube was not agitated. 4. Balancer itself gives rise to embryonic lethality	1. Allow longer time for eggs to hatch, some strains might require a longer incubation. 2. Bleach for a maximum of four minutes. 3. Use at least 5 ml of M9, have worms in a 50 ml conical, and be sure they are rotating during the entire incubation. 4. Homozygous lethal balancers and reciprocal translocation balancers produce some inviable embryos. Some proportion of embryos will not hatch, and this should not disrupt further applications provided there are enough live L1's left for sorting.
21	Recurrent clogged nozzle on the FACS	1. Did not bleach stock well enough, leading to excess of debris 2. Filtering insufficient 3. Worm suspension too concentrated	1. Bleach for a longer amount of time to reduce debris from adult worm bodies. 2. Dilute worm solution more before filtering or filter an additional time. 3. Dilute worm suspension.
22	No clearly distinct GFP- negative population visible	1. Samples with high debris content will have more debris that passes through the FSC vs SSC worm gate (Figure 4c left panels). Such samples will display events the GFP vs. RSA plot with a wide range of autofluorescent intensity, visible as a diagonal smear obscuring the GFP-negative population (Figure 4c right panels)	1. Use the upper and lower limits of RSA- intensity of the GFP-positive gate to guide the placement of the GFP-negative gate (Figure 4c right panels).
25	Collected GFP- larvae do not have desired genotype	1. Balancer unstable	1. Check GFP- animals from balanced stock to determine if they have the desired genotype. If not, the balancer may have broken down and permitted recombination. Thaw a frozen stock of the strain to recover the original balanced line. If this is a recurrent problem, then try a different balancer.
25	Some inviable larvae recovered	1. Reciprocal translocation balancer used	1. In GFP-marked reciprocal translocation balancers the GFP insertion is present on just one of the translocated chromosome arms. Depending on the location of the terminal allele, there could be GFP- half-translocation (aneuploid) animals that are not homozygous for the terminal allele. If the recovered animals are to be used for a chemical or genetic screen, then the presence of some GFP- inviable larvae of the wrong genotype should not disrupt the screen provided there are enough viable larvae collected. To avoid aneuploidy, use a balancer that is not a reciprocal translocation.