GSM6696581: YRR1, Calling Card Assay; Saccharomyces cerevisiae; OTHER
“Calling Cards” Assay: After TF directed transposition induction and selection, genomic DNA was extracted using a standard glass bead-beating, phenol chloroform isoamyl alcohol purification, and NH4Ac ethanol protocol. Genomic DNA was then divided into 3 separate enzyme digest reactions consisting of either HpaII, HinP1I, or Taqα1  Digested DNA was then, self-ligated using T4 DNA ligase, and Ty5 calling cards were recovered from genomic DNA in the self-ligated template by inverse PCR.  The desired yeast strain for each experiment is created by co-transforming two plasmids, one plasmid contains a Sir4p-tagged TF regulated by the ADH1 promoter with a LEU2 and URA3 auxotrophic marker, and the second contains a Ty5 transposon driven by the inducible GAL1/10 promoter with a HIS3 auxotrophic marker.