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iGEM > Paris >  Protocols >  Microscope Protocol

Protocols suggested
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a.menu_sub_active { padding-left: 7px; padding-right: 7px; color:#b0310e; font-weight:bold; }  Main |  Microscope Protocols|  Adapted Protocols|  Culture Protocols|  Molecular biology

Bacteria culture

 * 1) Chose different colors beware incompatibility with GFP+YFP (green and yellow to close)
 * 2) Membrane bacteria coloraiton with DID

Blade preparation
(do this step a few hours before; the blade need to be dry)


 * 1) Clean the glass blade
 * 2) Put the small blade on it (the blue square)
 * 3) Prepare the 1,5% Agarose + LB, boil it (microwave), let it cooling down
 * 4) take 50 µL agarose, put it in the middle of the blue square on the blade.
 * 5) put on it another blade, beware do not make bubbles, press it until 30sec
 * 6) 10min at 4°C

Cells preparation

 * 1) Resuspend bactéria in 1mL LB in an ependorff tube
 * 2) Quick centrifugation (low RPM)

Blade

 * 1) Take back the blade from the fridge
 * 2) Pull off the upper blade (slide it)
 * 3) Cut exceeded Agarose (if we want multiple observation, cut few Agarose)
 * 4) Put cells on the agarose gel (1µL)
 * 5) dry it with air agitation
 * 6) put a little blade on the bigger one, beware of bubbles

Microscopy observation

 * 1) power on the computer (what a nice idear)
 * 2) light up the 4 switch starting from the right one
 * 3) light up the green one
 * 4) stay at 37°C
 * 5) use MetaMorph sotware
 * 6) put the blade
 * 7) adjuste (use oil if you use x100)
 * 8) choose "Trans No Filter" (white light)
 * 9) use Acquire->Acquire->More
 * 10) take care of camera temperature (need to stay at -30°C
 * 11) Show live (if no image verify the display position)
 * 12) Setting Modified (exposition settings)
 * 13) if " Trans No Filter" (at the begining): put "100 ms trans on"
 * 14) if filter (CFP, GFP, etc...): put "1s fluo at"
 * 15) if it's not lean yet: put "4s fluo at"
 * 16) Beware we didn't have all the filter (GFP for example). If we want the RFP filter, we need to take the last one...
 * 17) >Acquire for picture or >Show live
 * 18) When it's done
 * 19) Slide up the camera
 * 20) Use the joystick to take out the blade
 * 21) Clean the camera with kodak paper
 * 22) wait 30min for the 1st switch starting from the left

Time laps variant

 * 1) >Journal/edit journal
 * 2) >igem/journal/TLPS_phase.JNL
 * 3) Choose in the TEST file, edit filter, positions, time etc....
 * 4) Run journal on TEST, >stop