Paris/PROTOCOLS

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For newcomers in wetlab: see also the course by D. Endy [http://openwetware.org/wiki/20.109%28S07%29:Lab_tourlink Laboratory Fundamentals of Biological Engineering].

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?

Pipette RAININ.jpg
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 Culture liquide bact.jpg.
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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: dehydrated strain

Paris ampoule.jpeg

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

Low Nitrogen Minimum Medium

The LNMM medium contains:

  • 2g KH2PO4
  • 1.18g succinic acid
  • 0.1g NH4SO4
  • 980mL H2O
  • water adjudted to pH 7.0 with KOH


After autoclaving, 20mL of sterile MgSO4 2% (w/v) are added.
Solid media were prepared by the addition of 1.5% (w/v) agar-agar.
Culture were incubated at 30°C aerobic.

LNMM

Nile Red

Sigma (N3013)

The dye is used at 0.5µg / mL medium. (liquid or solid).

  • make a solution of 0.25mg Nile red / mL DMSO
  • take 0.002 mL/mL medium from the previous solution (final concentration 0.5µg Nile Red/ mL medium).

This dye is excited by light around 450-500 nm and the emission light is > 528 nm (red). It can be exited around 515-560 and emission light is >590.

Nile Red

Chemical transformation

This protocol is based on the Invitrogen protocol (Subcloning Efficiency Competent cells). It uses DH5alpha E.coli strain chimically competent.

  • Put 50µL bacteria into 1.5mL microcentrifuge tube and incubate within ice.
  • Add 1-5µL (1-10ng) of DNA to the cells and mix gently (do not mix by pipetting up and down!)
  • Incubate on ice for 30 minutes
  • Heat shock cells for 20 seconds in a 42°C water bath without shaking
  • Place tubes on ice for 2 minutes
  • Add 950µL of pre-warmed medium of choice to each tube
  • Incubate tubes at 37°C for one hour at 225rpm
  • Spread 20 to 200µL from each transformation on pre-warmed selective plate.
  • Store the remaining transformation reaction at 4°C
  • Incubate plates overnight at 37°C

Fluorescent single cells visualisation

Slide preparation

  • Make LB-agarose : 0.15g of agarose in 10ml LB
  • warm it up in the microwave (a lot!!, to avoid cristal of agarose that could remain in the gel => ugly over microscope)
  • 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
  • put a droplet on the gel:
  • if you have a solid culture:
- pick up some colonies
- suspend within 100µL liquid medium (M9)
- put 1-2 µL on the gel
  • if you have an avernight liquid culture:
- take 1mL of the culture
- centrifugate 1000 rpm 1 min
- resuspend into 150 µL of medium (M9)
- put 1-2 µL on the gel slide
  • 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 can put wax around

Turning on the microscope

Zeiss Axiovert 200M
  • turn on : the light, the microscope, the shutter, the joystick
  • launch Metamorph
  • turn up the condenser and the objectives in load position
  •  !! take care : not keeping the light on if we doesn't use it
  •  !! do not switch on short after
  • wait 5 min (the microscope is checking the ranges of movement

Visualisation

Preparation

  • put a droplet of immersion oil on your slide, and attach it on the slide
  • Journal >> Show task bar
Metamorph-Taskbar.JPG
  • Task bar
>> Trans ON/OFF: turn on the halogen lamp
>> Binocular/Camera: turn on the binocular or the camera
>> Choose the fiter
  • Move up the objective until oil touches the slides
  • Turn on the binocular with trans filter
  • Move up the stage with micrometric "screw" until you see your cells, or something moving
  • Acquire:
- Digitizer (10MHz!!)
- Image >> New !!! ( without this, each image you take will overwrite)
- Settings >> (trans ou fluo; choice of exposure time)
Metamorph-acquire.JPG
  • Turn on the camera (Task bar >> Camera)


Trans Image

Turn trans light on (Task bar >> Trans ON) and trans filter
Acquire >> 100ms trans
Acquire >> Show Live
Acquire >> Acquire: acquire the trans image


Fluo image

Task bar >> fluo filter; trans OFFOFF)
Acquire >> 1s fluo
Acquire >> Acquire: acquire the fluo image


  • Acquire your images, etc, have fun
  • save your images in E:\iGEM\YYYY-MM-DD\a_proper_name


Metamorph.JPG

Turning off the microscope

  • Turn off the software, the microscope and the differents machines

NB : Nile Red, choose mRFP1/Texas Red filter


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