By L.Douglas Smoot;Philip J. Smith
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Sterilize seeds. 5–2 ml) or a 15 ml polypropylene tube depending on quantity and suspected level of contamination of seeds. The more seeds there are (or the more contamination there is) the larger the volume should be. 2 ml volumes for each step. For 15 ml tubes use 2–3 ml volumes for each step. 05 % Triton-X-100 for 10 min. (c) Remove EtOH. 05 % Triton-X-100 for 15 min. (e) Remove bleach and rinse five times in ½ MS. 48 Colleen Knoth and Thomas Eulgem Fig. 1 Scheme illustrating screening procedure.
The many benefits this strategy presents, such as a high level of temporal, reversible control over a chemical’s target protein, result in a highly effective approach towards unraveling complex and dynamic plant processes. Once an interesting molecule has been identified through chemical screening and its effects characterized, reverse genetics can then greatly assist in dissecting the precise actions of the chemical. Arabidopsis thaliana is an ideal tool for reverse genetics studies; a large and diverse abundance of mutants of this model species are widely and cheaply available to the research community.
For large-scale experiments measuring multiple microplates in series with the TopCount instrument, seal plates with foil and pierce holes into it to avoid condensation, which may impair luminescence detection. For small-scale assays using single microplates in a simple luminometer sealing is not required. 17. Initial luciferase activity is proportional to the amount of proteins expressed from the activated reporter gene (and the turnover of the protein). However, since luciferase is Luciferase-Based Screen for Bioactive Compounds 29 inhibited by its product oxyluciferin, maximum enzyme activity will decline over time and reach a steady state after a certain time.