User:Emmalee Jones/Notebook/Lab Notebook/2010/03/30

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Polymerizing MTs and Flow Cells
Polymerize MT
 * Turn on thermocycler machine
 * If not already out, get Antifade from freezer and thaw
 * Get MT from big freezer (Fluorescine labeled) - then stick in thermocycler for 30 minutes
 * Dilute Taxol from 10mM to 10micromolar to make PEM-T (make 500 microliters so .5 microliters of the 10mM to 499.5 of PEM) - must use within 3 hours
 * When MTs done in thermocycler, add 199 microliters from PEM-T to MTs while in cycler, then wrap with foil to preserve fluorescence
 * Take out a flow cell - rinse the lysine coated surface 3 times with PEM

Put in motility solution (10 microliters)

Motility Solution
 * 91.5 microL PEM-T
 * 2.5 microL AF
 * 1 microL PEM-Glu
 * 5 microL MT (the ones I just polymerized)
 * Let sit for 10 minutes in drawer


 * Take out and rinse three times again with PEM -T

Now add the lipid solution (10 microliters) and let sit in drawer for another 10 minutes Lipid Solution
 * 50 microL Lipids
 * 2.5 microL AF
 * 1 microL PEM-Glu
 * 46.5 microL PEM
 * Rinse three times with PEM-T again, then seal with nail polish on ends of the flow cell

Notes on microscope

 * First thing is to focus objective on tape on flow cell
 * Then focus the edge of the stop (at top of microscope), want to get a clean edge on it, and still have a large enough circle of light to cover all the area you can see - use the large knob at the back to focus, use the little tiny knobs on the front to move the area that the light covers.
 * For the 60x objective need to do it in oil - take out slide, put oil straight on the objective glass surface at the top, put in slide.
 * Looks like MTs didn't stick. In fact, looks like almost nothing stuck.  Hmmm.  Will try another of these flow cells tomorrow.

Other notes:
 * The Antifade smells terrible. Be careful and fast when using.
 * I should start making the lipids in PEM instead of in PBS.