cDNA library screening

A unidirectional cDNA library, against mature male kidney cDNA, constructed in Lambda ZAP Express?(Stratagene, La Jolla, CA, USA) was used to screen for AR cDNA using a DIG-labeled AR cRNA probe. The screening was conducted according to the Lambda ZAP Express?manual (Stratagene). DIG labeled anti-sense RNA probes were generated using the DIG RNA Labeling Kit (Roche, Mannheim, Germany). Hybridization was performed at 45癈 overnight (O/N) in hybridization buffer (5 ?SSC, 50% formamid, 0.02% SDS (w/v), 0,1% N-laurylsarcosine (w/v) and 2% blocking solution (w/v)) (Roche). Membranes were washed for 2 ?5 min in 2 ?SSC and 0.1% (w/v) SDS at room temperature, and for 2 ?15 min in 0.2 ?SSC, 0.1% (w/v) SDS at 68癈. Signals were detected using CSPD (Roche) and exposure of Hyperfilm?MP film (Amersham Pharmacia Biotech, Buckinghamshire, England) and hybridization signals were visualized using a CURIX 60 Film Developer (AGFA-Gevaert AB, Kista, Sweden). Positive plaques were purified through four successive hybridization rounds, and individual clones were isolated by phagemid excision. Following sequence identification of the clones as AR, they were sequenced to completion by Cybergene AB (Huddinge, Sweden).