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Protein Phenotype Testing & Colony SDS-PAGE Analysis

Explore colony PCR results, extract proteins, conduct SDS-PAGE gel electrophoresis, perform yeast phenotype testing, and plan for future experiments. Includes steps for whole cell extraction, blotting, and spotting tests. 8

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Protein Phenotype Testing & Colony SDS-PAGE Analysis

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  1. Phenotype Testing and SDS-PAGE Mod2 Day4 Thursday Oct 16th, 2008

  2. First let’s look at colony PCR results

  3. SDS-PAGE • You will make a whole cell extract from equal # of cells --> OD600 • Glass beads/SDS/BME/boiling steps • Gel separates proteins based on size • May want to change lane order for + control • After 1 hour run, blot to nitrocellulose and block with milk solution until next week.

  4. Yeast Phenotype Testing • Choose plates you will use and put them in the incubator to dry out before running your protein gel • Spot test demo…

  5. For next time… • What size is the protein you’re looking for (TAP = +20KDa) • Make a figure of colony PCR result • Calculate number of cells for RNA extraction

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