215-Lab10A-Yeast_Tsf

215-Lab10A-Yeast_Tsf - Lab 10A Yeast Transformations Lab...

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Lab 10A – Yeast Transformations 10A-1 Lab 10A: Yeast Transformation In the previous experiments you assayed for the ability of the Hst1::Sir2 chimera to silence expression of a TRP1 reporter gene. This assay is used to determine if the chimera has Sir2 silencer activity. In the next series of experiments we need to determine if the mutant chimera retains Hst1 activity by assaying its ability to repress transcription of a LacZ reporter promoter under the control of an MSE. To perform the repression assays you will need to transform the mutant chimera plasmid into strain JXY5 ( ura3 , leu2 , hst1 ) that contains a MSE regulated LacZ reporter on plasmid pJX43 ( URA3 ). Cells that are transformed with the mutant Hst1::Sir2 chimera plasmid pJM477 ( LEU2 ) will be selected for their ability to grow on SD – ura leu. In the following weeks we will test if the chimera is able to repress the LacZ reporter. 1. Label two
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215-Lab10A-Yeast_Tsf - Lab 10A Yeast Transformations Lab...

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