Paris/PROTOCOLS
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Nicolas C. (Talk | contribs) (→Titration of bacteriophages P1) |
Nicolas C. (Talk | contribs) |
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*add 1.9mL of erythromycin (stored in the freezer at 133x) | *add 1.9mL of erythromycin (stored in the freezer at 133x) | ||
*spread the medium in about 10 petri dish | *spread the medium in about 10 petri dish | ||
| + | |||
| + | == Microscopy == | ||
| + | |||
| + | === Fluorescent single cells visualisation === | ||
| + | * Slide preparation | ||
| + | ** Make LB-agarose : 0.15g of agarose in 10ml LB | ||
| + | ** warm it up | ||
| + | ** wash the slide (special slide with "two holes" with ethanol) | ||
| + | ** put ~80µL of LB-agarose in each hole | ||
| + | ** spread the gel with another slide and wait for ~2 min | ||
| + | ** remove extra gel with a cutter | ||
| + | ** coverslip/nailpolish | ||
| + | ** depending on the concentration of your culture, put a droplet (1.5µL) on the gel | ||
| + | ** spread it by moving the slide | ||
| + | ** wait until you don't see the droplet anymore (2min) | ||
| + | ** put a coverslip and attach it with nailpolish | ||
| + | ** optionally : you | ||
| + | |||
[[Paris|<<home]] | [[Paris|<<home]] | ||
Revision as of 09:04, 13 July 2007
Contents |
Getting started
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 
.
<<home
Strains
Here you can find the list of strains we have.
E. Coli MG1655
WT
E. Coli w121
We got the w121 strain from a lab in Pasteur Institute. This strain is [DapA-; Erythromycin R], but also has a couple of other mutations we are not interested in.
E. Coli Ftsz -TS84
Three clones are available : 121.1, 121.2, 121.3. More details soon
Acinetobacter
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...
Titration of bacteriophages
- Take your stock of bacteriophage
- make several dilution of your stock, for example :
- 10µL of stock in 990µL MgSO4 0.1M -> d2
- 10µL of former solution in 990µL MgSO4 0.1M ->d4
- 10µL of former solution in 990µL MgSO4 0.1M ->d6
- 10µL of former solution in 990µL MgSO4 0.1M ->d8
- In a petri dish containing LB, spread a solution of 100µL of E. Coli MG1655 in stationnary phase + warm top agar 900µL (TA,7)
- Spread this mix over a petri dish
- add droplets (7µL) of your phages dilution on the petri dish, mark the places where you put these droplets
- Incubate ON at 37°C
- check for lyse plaque
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
Microscopy
Fluorescent single cells visualisation
- Slide preparation
- Make LB-agarose : 0.15g of agarose in 10ml LB
- warm it up
- wash the slide (special slide with "two holes" with ethanol)
- put ~80µL of LB-agarose in each hole
- spread the gel with another slide and wait for ~2 min
- remove extra gel with a cutter
- coverslip/nailpolish
- depending on the concentration of your culture, put a droplet (1.5µL) on the gel
- spread it by moving the slide
- wait until you don't see the droplet anymore (2min)
- put a coverslip and attach it with nailpolish
- optionally : you
