IGEM:IMPERIAL/2008/Prototype/Wetlab/Protocols

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<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype><img width=50px src=http://openwetware.org/images/f/f2/Imperial_2008_Logo.png></img</a></html> Home The Project B.subtilis Chassis Wet Lab Dry Lab Notebook

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Motility Experiments

Isolating motile derivatives

  1. Streak each strain carefully at one point of a 0.7% TBAB agar plate.
  2. Incubate overnight at 37ºC without inverting the plates.
  3. Motile cells will swarm over the surface of the plate and should be picked from as far from the site of inoculation as possible. If the strain was non-motile there will be a contained streak with a few dendritic swarms emerging from the streak. Pick from the top of one of these swarms to recover a motile derivative.

Microscope Protocol

(For GFP Microscopy)

  1. Grow cells overnight at 22°C in LB to 1 OD_600
  2. Pellet 1ml and resuspend in 0.1ml PBS
  3. Stain membranes by resuspension in 50μL of PBS containing 5μg/mL FM4-64 and incubate for 10min at room temperature.
  4. Observe samples by spotting 3μL of suspension on a glass microscope slide and immobilise with poly-L-lysine-treated glass coverslip.
  5. Capture images


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