Schematic outline of the mutant C.elegans library generation and screening. (A) Library generation: P0 worms were treated with EMS and F1 progeny were singled out and expanded clonally until the F2 to F3 generation. Cultures were split in two, where the first half was used for cryopreservation in duplicate and the other half for DNA isolation and subsequent mutation detection in genes of interest. (B) Library screening: Genic regions of interest were amplified by a nested PCR and screened for induced mutations by dideoxy resequencing. Mutations were scored by PolyPhred 5.0 (Nickerson et al. 1997) and stored and annotated in LIMSTILL (http://limstill.niob.knaw.nl; V. Guryev and E. Cullen, unpubl.). When an interesting mutation was identified, it was reconfirmed in an independent assay and the mutant was retrieved from the frozen archive, outcrossed, and analyzed for phenotypic consequences. (C) Nested PCR setup: In the first PCR multiple regions of interest are amplified in a multiplex of various oligo 1 and 4 combinations. The second PCR was performed with a single pair of M13-tailed oligos 2 and 3. The resulting products were sequenced using universal M13F and/or M13R oligonucleotides.
