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Comprehensive Lab Protocol: Western Blot, β-Gal Assay, and Sequence Analysis for Mutants

This detailed lab protocol is divided into three parts: 1) Conducting a Western blot to evaluate protein expression, including the rationale behind the procedure. 2) Performing a β-galactosidase assay (Miller Units) to assess the activity of your mutants in both light and dark conditions, with considerations for experimental replicates and tagging. 3) Engaging in sequence analysis where you retrieve sequences, determine reverse complements, and conduct a BLAST search against pCph8, assessing potential HA tag inclusion and implications of amino acid changes.

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Comprehensive Lab Protocol: Western Blot, β-Gal Assay, and Sequence Analysis for Mutants

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Presentation Transcript

  1. System Engineering M2D3 11.03.09

  2. Three part lab today Part 1: finish Western Part 2: b-gal assay for your mutants Part 3: sequence analysis

  3. Part 1: finish Western Why are we performing a Western blot?

  4. Part 2: b-gal assay Miller units Why are we testing both light and dark grown cells? Should we test + and – HA tag? How many replicates should we test?

  5. Part 3: Sequence analysis Retrieve the sequences find the reverse complement BLAST your sequence (= query) vs pCph8 (= subject) Will pCph8 have HA tag? What will you do with your amino acid changes (if you have any)?

  6. Part 3: Sequence analysis

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