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The transformants were plated on SCM-Ura medium to select for clones that have successfully assembled the plasmid.
Mixed cultures of K76A/pLhyS-2 K-Pa1 and K9AL/pAUR112 were plated on YPD + HYG, AUR medium to select for hybrid K76x9/pLhyS-2 K76x9/pLhyS-2 cells, and all plasmids were then removed from the hybrid cells via cultivation in YPD medium without antibiotics to yield multi-hybrid strain K76x9 (Table 1).
After 48 hr the whole colonies were resuspended and plated onto a defined medium to select for Ade+ cells.
The liquid library was inoculated into challenging medium to select for mutants of higher tolerances toward different stresses.
Blasticidin (2.5 μg/ml) was added to the culture medium to select for transformed parasites.
These strains were grown in SC-lys-tyr medium to select for the disomes and 10−2, 10−3, and 10−4 dilutions were plated on YPD + cycloheximide medium to select for loss of heterozygosity for the cyh2 allele.
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The next day the medium was removed, cells were rinsed with DMEM, and TG or AzHx selective medium was to select against or for HPRT expression, respectively.
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For bottom-up in vitro SCRaMbLE, SC Ura His medium or SC His + 5-FOA medium are used to select for recombined constructs, depending whether the first or second version is used.
The library was then successively spotted on Edinburgh minimal medium (EMM; Formedium) to select for prototrophs and on YES medium with G418 to select for the kanMX4 cassette used for generating deletions.
Approximately 1×109 cells were plated to medium lacking histidine to select for translocations, and dilutions plated to YPD medium to determine viability.
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