PMC:329117 (10) JSONTXT < >
|Materials and methods|
|Identification of olfactory receptor cDNAs|
An adult mouse cDNA library made from the olfactory epithelium of a single animal was provided by Leslie Vosshall (Rockefeller University, New York, NY, USA), and an embryonic library (made from the olfactory epithelia of several E16.5-E18.5 embryos) was provided by Tyler Cutforth (Columbia University, New York, NY, USA). Both libraries were oligo-dT primed and directionally cloned into the lambdaZAP-XR vector (Stratagene, La Jolla, CA, USA). The adult library has a complexity of 6.5 × 106 primary clones, and the embryonic library has a complexity of 1.65 × 106. Libraries were amplified to give titers of 5 × 109 pfu/ml (adult) or 2 × 1010 pfu/ml (embryonic). Hybridization probes were made by degenerate PCR of mouse genomic DNA, in a fashion similar to those described previously , with primer pairs and annealing temperatures given in Table 2. Low-stringency hybridization conditions were as described . 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. Plasmid DNA gave better 3'-end sequence than PCR products, which often suffered from polymerase stuttering through the poly(A) tract.
cDNA sequences and associated information are available through dbEST (Genbank accessions CB172832-CB174569) and our olfactory receptor database . The updated olfactory receptor gene catalog is available through Genbank (accessions AY317244-AY318733). Throughout the manuscript, genes are referred to by their Genbank accession numbers.
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