Strain construction and microbial techniques
This protocol is extracted from research article:
Structural basis of Stu2 recruitment to yeast kinetochores
eLife, Feb 16, 2021; DOI: 10.7554/eLife.65389

Saccharomyces cerevisiae strains used in this study, all derivatives of M3 (W303), are described in Supplementary file 1. Standard media and microbial techniques were used (Sherman et al., 1974). Yeast strains were constructed by standard genetic techniques. Construction of pCUP1-GFP-LacI and ipl1-321 are described in Biggins et al., 1999, CEN3::lacO:TRP1 is described in Shonn et al., 2003, mad3Δ in Pinsky et al., 2006, DSN1-6His-3Flag in Akiyoshi et al., 2010, stu2-3V5-IAA7 in Miller et al., 2016, and TOR1-1, fpr1∆, and MPS1-FRB:KanMX in Aravamudhan et al., 2015; Haruki et al., 2008. STU2-FRB:HisMX and NUF2-FKBP12:HisMX were constructed by PCR-based methods (Longtine et al., 1998). Strains containing the previously described pMET-CDC20 allele were provided by Frank Uhlmann. pGPD1-TIR1 integration plasmids (pM76 for integration at HIS3 or pM78 for integration at TRP1) were provided by Leon Chan. Construction of a 3HA-IAA7 tagging plasmid (pM69) was described previously (Miller et al., 2016). Construction of a LEU2 integrating plasmid containing wild-type pSTU2-STU2-3V5 (pM225) and pSTU2-stu2Δ855–888-3V5 (pM267) are described in Miller et al., 2016; Miller et al., 2019. STU2 variants were constructed by mutagenizing pM225 as described in Liu and Naismith, 2008; Tseng et al., 2008. Primers used in the construction of the above plasmids are listed in Supplementary file 2, and further details of plasmid construction including plasmid maps are available upon request.

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