Complementation in liquid media

Daniel Muncanovic; Mette Heberg Justesen; Sarah Spruce Preisler; Per Amstrup Pedersen

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Complementation in liquid media

DM Daniel Muncanovic

MJ Mette Heberg Justesen

SP Sarah Spruce Preisler

PP Per Amstrup Pedersen

This method is extracted from research article: Sci Rep, Aug 2019

Characterization of Hailey-Hailey Disease-mutants in presence and absence of wild type SPCA1 using Saccharomyces cerevisiae as model organism

DOI: 10.1038/s41598-019-48866-y

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Yeast cells were cultured from −80 °C stock as above. Complementation assays were performed in clear 96-well plates (well capacity: 400 µl). Each well contained 200 µl yeast culture. Medium was made from 100 µl 2x SG medium and 80 µl of CaCl₂, BAPTA or MnSO₄ (all dissolved in 100 mM TES/KOH, pH = 6) to the desired final concentrations. 20 µl of a cell suspension with OD₄₅₀ = 0.5 in dH₂O was added to each well. Plates were incubated at the desired temperature and OD₄₅₀ of the cultures measured in a plate-reader (Multiskan RC, Thermo Labsystems) at appropriate time intervals. BAPTA (1, 2-bis(o-Aminophenoxy)ethane-N,N,N’,N’-tetraacetic Acid) was purchased as tetrasodium salt from Merck (cat.no.: 196418).

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