Paris/PROTOCOLS
From 2007.igem.org
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Prepare to begin
This topic is adressed to all our informatics-physics-I'm-afraid-of-the-bench fellows. So, if you finally found the courage to dare the pipettes, PCRs and nicely smelling bacteria, welcome!
- What a pipette is?
Pipettes dispense various volumes. The plunger button indicates the maximum volume (microliters) that the pipette is designed to handle. For example, P-20 will handle up to 20 microliters.
The digital volume indicator is read from top to bottom. For P-2, P-10, P-20, P-100, and P-200, black digits indicate microliters and red digits tenths and hundredths of microliters. For P-1000, red digits indicate milliliters and black digits microliters.
What to do when you have it in you hand?
-Hold the pipette in one hand (it doesn't bite...). With the other hand, turn the volume adjustment knob counterclockwise so the volume indicator is 1/3 revolution above the desired setting, then slowly turn clockwise until the indicator shows the desired volume.
-Attach a new disposable tip to the pipette shaft.
-Press the plunger to the FIRST stop. This part of the stroke is the volume displayed by the indicator.
-Holding the pipette vertically, immerse the tip a few millimeters into the sample.
-Allow the pushbutton to return slowly to the UP position. Avoid to blurt out the plunger button abruptly : there are bulls appearing and your volume is false...
-Ensure that the full volume of sample was properly drawn into the tip.
-Withdraw the tip from the sample.
-To dispense the liquid, gently touch the tip to the side of the receiving vessel, immersing the tip into liquid within the vessel. Press the plunger to the SECOND stop.
-With the plunger fully pressed, withdraw the tip carefully, wiping residual drops against the vessel wall.
-Allow plunger to return to the UP position.
-Discard the tip by depressing the tip ejector button.
Note down that different tips exist : ensure that you have the right one (labels will indicate you the size, etc.). It's better to use filter tips.
To train, you can simply pipette water : it's important to know how much 1 µl is...
To be continued...
- Growing bacteria in liquid medium
-Light the Bunsen burner. It permits you to keep a 10 cm perimeter sterile et thus not to contaminate your future colonies.
-Get a 50mL Falcon tube and put into 5 mL of LB medium. Add supplementary stuff if needed (antibiotics, metabolites, etc.).
-Pick up a sterile toothpick. Use it to gather a single colony of cells (you know, a white point on your Petri dish...).
-Place the toothpick with the colony into the solution.
-Incubate overnight at 37°C with shaking (at about 200 rpm).
Next morning, after a cup of coffee and a croissant, you can check up 
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Transduction with P1 bacteriophage
Preparation of the P1 stock on the w121 strain.
Step of Tuesday, July 3
To be completed...
Transduction to MG1655 using the P1 stock made on w121.
Step of Wednesday, July 4
To be completed...
Preparation of DAP solution from the powder (50mM)
Step of Friday, July 6
- M(DAP)=190.2g/mol
- I put 0.285g of DAP in 30ml water
- Aliquoted by 15ml
- Stored in the freezer at -20°C
- the stock is 166x
Preparing growth media
Making 10 petri dish (LB+tet+citrate+DAP)
- take 250ml of LB
- warm it up in the microwave for ~ 6min
- wait until you can handle the bottle for 2sec
- add 5ml of citrate 1M
- add 1.5ml of DAP
- add 250µL of tetracycline (stored in freezer at 1000x)
- spread the medium in about 10 petri dish
Making 10 petri dish (LB+erythromycin+citrate+DAP)
- take 250ml of LB
- warm it up in the microwave for ~ 6min
- wait until you can handle the bottle for 2sec
- add 5ml of citrate 1M
- add 1.5ml of DAP
- add 1.9mL of erythromycin (stored in the freezer at 133x)
- spread the medium in about 10 petri dish
