Figure 1 .

PCR/deletion screening. Worms were grown in liquid culture in arrays of 96. A portion of each well was harvested for DNA preparation while the rest was frozen for later retrieval of animals. DNA was pooled into a grid of rows and columns, which could later be used to address individual wells. Nested PCR primers were designed for amplification of target region. Primers were designed using the program Aceprimer (McKay and Jones 2002). Shown are four slab gels stained with SYBR Gold used to identify deletions (faster running bands) from the wild-type DNA (slower bands). The four panels are (A) the initial screen, (B) first sib, (C) second sib, and (D) third sib. The WT band for this primer set is 2099 bp (long arrow), and the marker is a 100-bp molecular weight ladder with strong intensity bands at 1 kb and 3 kb (marked by horizontal bars on right hand side of image). The screening image (A) shows two different hits (in duplicate screening, short arrows), one at just over 1 kb (18^th set of four lanes, in first two lanes of the four), and one at about 1.6 kb (23^rd set of four lanes, in first two lanes). The one at 1 kb passed addressing but was not recovered in sib selection, so the remaining images (B–D) are for the 1.6 kb candidate. Through rounds of sib selection, one enriches for animals segregating the deletion band. This enrichment proceeds from initial detection in (A), a mix of hundreds to thousands of animals, to first-round sibbing in (B), tens of animals, and finally single animal picks in sib2 (C) and sib3 (D), where one has single animals segregating the deletion band. For sib3 (D), there are 4 × 24 single-worm populations, 1–4. Populations 1 and 2 are in the first half of the comb, and 3 and 4 are in the second half. Set 3 is actually 24/24 positive, whereas the others are less than that, so set 3 was picked to go forward as it was homozygous at that point. The example shown is gk3287 in the gene F11E6.1 (gba-3).