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  MoBY-ORF Clone Construction

Each plasmid in the MoBY-ORF library was constructed in a three-step process.

First, using the primers listed in Supplementary Table 1 and Table 2, each yeast ORF was PCR-amplified from DNA isolated from the sequenced S288C strain (1 May 2005 freeze). A DNA fragment encompassing on average ~900bp upstream of start codon and ~250bp downstream of stop codon (unless the intergenic region is less than ~250bp) of each ORF was PCR-amplified. The KanMX barcode sequences were PCR-amplified from the deletion mutant corresponding to the amplified ORF.

Second, to facilitate cloning by homologous recombination, the ORF and barcode PCR products were co-transformed into yeast, along with XhoI-linearized p5472 (Supplementary Figure 1).

Third, recombinant plasmids were recovered in bacteria to facilitate plasmid DNA isolation and subsequent diagnostic restriction digests to confirm the sizes of both fragments (Supplementary Figure 1).

Bacterial host strain: BUN20 [∆lac-169 rpoS(Am) robA1 creC510 hsdR514uidA(MluI):pir-116 endA(BT333) recA1 F’(lac+ pro+ ∆oriT:tet)] (Li and Elledge, 2005)

Bacterial origin of replication: ori6Kγ (therefore, MoBY-ORF plasmids can only be replicated in a pir-116 host i.e. NOT DH5α)

Bacterial selectable markers: chloramphenicol, kanamycin (grow bacterial strains in presence of BOTH drugs)

Yeast origin of replication: CEN/ARS

Yeast selectable markers: G418, URA3

Supplementary Figure 1

Copyright © Boone Research Laboratory at CCBR, University of Toronto, 2009