Rapid isolation of genes from an indexed genomic library of C-cinereus in a novel pab1(+) cosmid

In this study we present an indexed genomic library of homokaryon AmutBmut constructed within a novel cosmid carrying pab1(+) as a selectable Coprinus marker. The average insert size per cosmid comprises 41 kb. We screened th e library and detected copies of known (a1-2, beta-tub, cgl1, ras, trp1) an d of new Coprinus genes (cac, lac1, lac2, lac3). Screening was performed ei ther by Southern blot hybridisation or more efficiently by non-radioactive PCR amplification. We successfully applied PCR with specific and with degen erate primers, multiplex; PCR and colony PCR in library screening. Our resu lts suggest a new, more efficient pooling strategy for future high throughp ut screenings to be used in PCR with pooled cosmid DNAs, or in a less labor ious approach using pooled Escherichia coli colonies for PCR. (C) 1999 Else vier Science B.V. All rights reserved.