Team:Paris/ProtocolsA

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Contents 1 Protocols suggested 1.1 Bacteria culture 1.2 Blade preparation 1.3 Cells preparation 1.4 Blade 1.5 Microscopy observation 1.6 Time laps variant

Protocols suggested

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)
    1. if " Trans No Filter" (at the begining): put "100 ms trans on"
    2. if filter (CFP, GFP, etc...): put "1s fluo at"
    3. if it's not lean yet: put "4s fluo at"
    4. Beware we didn't have all the filter (GFP for example). If we want the RFP filter, we need to take the last one...
13. >Acquire for picture or >Show live
14. When it's done
    1. Slide up the camera
    2. Use the joystick to take out the blade
    3. Clean the camera with kodak paper
    4. 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