Project Director: John Colbourne

A cosmid library - which contains DNA inserts of 35-40 kb - was created to accelerate gene capture by PCR screening and to allow the characterization of entire genes, including non-coding transcriptional regulatory regions. Given Daphnia's haploid genome size of ~300 Mb and the average cosmid cloning capacity of ~36 kb, a library of 30,000 bacterial clones achieves 95% probability of including a given gene of interest. Surpassing this mark, isolated cosmid-containing bacterial clones are arrayed into 100 384-well microtiter plates and archived at -80°C. DNA from each plate is pooled into a single well within 96-well plates. One PCR using gene specific primers identifies the 384-well plates containing the gene of interest. DNA from these plates, in turn, are pooled by column and row (16 x 24). Therefore, an additional PCR identifies the complete address of positive cosmids, which are then cultured, subcloned and sequenced.

Figure 1: PCR screening of pooled DNA reveals three 384-well plates (18,37,83) that contain a gene of interest.	Figure 2: An additional PCR of pooled DNA from columns and rows identifies the full address of the gene within the cosmid library (plate 18, column 10, row 8).

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