User:Emmalee Jones/Notebook/Lab Notebook/2010/02/15

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Lysine to Fix MTs in Place

To test how poly-L lysine fixed microtubules to a glass slide we first polymerized fluorescine labeled MTs following the procedure on Andy's MT page. The solution of MTs was:

  • 2.5 μL AntiFade
  • 1 μL glucose
  • 5 μL MTs
  • .1 μL taxol
  • 92.4 μL PEM

We then used a 5 mg/mL solution of poly-L lysine in PEM buffer, put 10 μL on a slide, waited 10 minutes (while MTs were finishing baking) then rinsed with 30 μL of straight PEM buffer.

After rinsing, we put the MTs on the slide. To prepare the microscope, added cedar oil (wash the cap after using) on the stage, then put slide on stage. To remember about the microscope: switch for mercury lamp below table (let warm up before using microscope), switch for camera next to the monitor, switch for microscope light on base of scope and switch between filters on bottom right of scope.

The microscope is run using a Labview program on the computer. To focus on the sample, first focus on the tape on the slide. The images mostly showed clumps of MTs that were not moving at all. They photobleached very quickly. Below are images from MTs that were not clumped.

LysineFlMTs.jpg

LysineFlMTs1.jpg