User:Steven J. Koch/Notebook/Kochlab/2009/05/26/FITC Microtubules with antifade

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Steve Koch 18:18, 26 May 2009 (EDT): Brigette, Larry, Anthony, Andy, and I succeeded in making FITC labeled MTs and visualizing them in antifade buffer. This was Brigette's first day in the lab and Larry's first time with MTs, so it's a very good omen that things were working very well.

FITC MTs

We took out one 2 microliter aliquot of FITC tubulin from last week. Polymerized MTs for 20 minutes using the thermal cycler at 37C with hotlid. Stabilized by adding 198 ul of room temperature BRB80-T

Antifade cocktail

Steve Koch 18:32, 26 May 2009 (EDT): For this, we used some old frozen components:

  • 10 microliters of Glucose oxidase (GOD) from last summer
  • 10 microliters of Catalase (CAT) from last summer
  • 5 microliters of an aliquot of beta-mercaptoethanol (BME) frozen at -20C from last summer.

Brigette mixed up this for a total of 25 ul of antifade cocktail.

Microtubules in antifade viewing solution

  • 85 microliters of BRB80T
  • 10 microliters of polymerized MTs
  • 2.5 microliters of AF cocktail
  • 2 microliters of "1.6 M Dextrose"
    • We made the dextrose in a very rough manner, just weighing out some powder and then adding in a volume based on the printed lines on the 50 mL centrifuge tube.

Visualization

Steve Koch 18:41, 26 May 2009 (EDT): To visualize, we just put a 5 microliter drop onto a coverglass and then put the coverglass on a slide. We marked the coverglass with a sharpie to make it easy to find the sample plane.

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