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.