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 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.
Bottoli
A P
AP
Mikrobiologisches Institut, ETH Zürich, Switzerland.
Kertesz-Chaloupková
K
K
Boulianne
R P
RP
Granado
J D
JD
Aebi
M
M
Kües
U
U
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D003302:Coprinus / Q000235:genetics*; D003360:Cosmids / Q000235:genetics*; D005800:Genes, Fungal; D015698:Genomic Library; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods