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.
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