PMC:329117 / 38693-40345 JSONTXT

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Hybridization probes were made by degenerate PCR of mouse genomic DNA, in a fashion similar to those described previously [1], with primer pairs and annealing temperatures given in Table 2. Low-stringency hybridization conditions were as described [1]. Clonally-pure plaques were obtained through secondary screens using the same probe as the corresponding primary screen. PCR with vector primers (M13F/R) was performed to prepare sequencing templates. cDNA size estimates were obtained by agarose gel electrophoresis, and inserts were sequenced from the 5' end using the M13R primer and big-dye terminator chemistry according to ABI's protocols (Applied Biosystems, Foster City, CA, USA). In order to obtain 3' sequence, selected phage clones were converted to plasmid stocks following a scaled-down version of Stratagene's in vivo excision protocol. 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