From 2007.igem.org

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 1 Getting started 2 Strains 2.1 E. Coli MG1655 2.2 E. Coli w121 2.3 E. Coli Ftsz -TS84 2.4 Acinetobacter: dehydrated strain 3 Transduction with P1 bacteriophage 3.1 Preparation of the P1 stock on the w121 strain. 3.2 Transduction to MG1655 using the P1 stock made on w121. 3.3 Titration of bacteriophages 4 Preparation of DAP solution from the powder (50mM) 5 Preparing growth media 5.1 Making 10 petri dish (LB+tet+citrate+DAP) 5.2 Making 10 petri dish (LB+erythromycin+citrate+DAP) 5.3 Low Nitrogen Minimum Medium 5.4 Nile Red 6 Chemical transformation 7 Glycerol Stock 8 Fluorescent single cells visualisation 8.1 Slide preparation 8.2 Turning on the microscope 8.3 Visualisation 8.4 Turning off the microscope

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 .
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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

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.

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.

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

Glycerol Stock

• add 1mL of 40% glycerol into cryogenic tube
• add 1mL of overnight bacterial culture
• vortex gently
• stock at -80°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

• 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

• 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)

• 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

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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