User:Brigette D. Black/Notebook/Brigettes Notebook/2009/05/26/Making Microtubules (+ Antifade)

Polymerizing Microtubules

Placed one aliquot of FITC tubulin (from -80 freezer) in the thermal cycler at 37 C for 20 minutes. Once thawed, we added 198 microliters of BRB80-T at room temperature.

Antifade Cocktail

• 10 microliters of glucose oxidase (GOD)
• 10 microliters of catalase (CAT)
• 5 microliters of beta-mercaptoethanol (BME)

• All components were stored in the freezer and thawed on ice

Antifade + Microtubules

• 85 microliters of BRB80-T
• 10 microliters of polymerized microtubules
• 2.5 microliters of AF cocktail
• 2 microliter of 1.6M dextrose

Viewing the Microtublues

We were able to look at the polymerized microtubules, first without then with the antifade solution by placing a 5 microliter drop of solution on a slide(marked with a sharpie), then sandwiched by a cover glass. The microtubules without antifade solution were visible but photobleached rapidly (5 seconds or so). After adding the antifade solution, the microtubules remained unbleached for much longer (>30 seconds).