Bottoli A P, Kertesz-Chaloupková K, Boulianne R P, Granado J D, Aebi M, Kües U
Mikrobiologisches Institut, ETH Zürich, Switzerland.
J Microbiol Methods. 1999 Mar;35(2):129-41. doi: 10.1016/s0167-7012(98)00109-2.
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 the library and detected copies of known (a1-2, beta-tub, cgl1, ras, trp1) and of new Coprinus genes (cac, lac1, lac2, lac3). Screening was performed either by Southern blot hybridisation or more efficiently by non-radioactive PCR amplification. We successfully applied PCR with specific and with degenerate primers, multiplex PCR and colony PCR in library screening. Our results suggest a new, more efficient pooling strategy for future high throughput screenings to be used in PCR with pooled cosmid DNAs, or in a less laborious approach using pooled Escherichia coli colonies for PCR.
