Figure_2.tif (1.04 MB)

Flow chart outlining the protocol used for split-ubiquitin-based screening of an Arabidopsis cDNA library for interactors of atToc159G, atToc132G and atToc132AG baits.

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posted on 2014-04-15, 03:16 authored by Siddhartha Dutta, Howard J. Teresinski, Matthew D. Smith

S. cerevisiae strain NMY51 was transformed with Toc159G, Toc132G or Toc132AG bait constructs and a split-ubiquitin yeast two-hybrid assay was performed utilizing positive, negative and empty prey plasmids. Optimization of the screening stringency was achieved through pilot screening, which involved large scale transformation of bait pre-transformed yeast with empty library vector. Selection of positive clones was conducted on the quadruple dropout media supplemented with 3-aminotrizole. The colonies that grew on the selective media were re-plated on the same stringent selective medium. Prey plasmids were re-isolated from the putative positive clones. Two degrees of selection (i.e. individual retransformation with respective baits and bait dependency test) were followed by sequencing and BLAST analysis to confirm the interactions and simultaneously identify the interactors.