From 2007.igem.org

Revision as of 09:20, 13 July 2007 (view source) Nicolas C. (Talk | contribs) (→Microscopy) ← Older edit		Revision as of 09:25, 13 July 2007 (view source) Nicolas C. (Talk | contribs) (→Visualisation) Newer edit →
Line 137:		Line 137:
	* put a droplet of immersion oil on your slide, and attach it on the slide		* put a droplet of immersion oil on your slide, and attach it on the slide
	* Journal >> Show task bar		* Journal >> Show task bar
-	* Trans ON/OFF : turn on the halogen lamp	+	* Task bar >> Trans ON/OFF : turn on the halogen lamp
	* Move the stage with the joystick to put the objective face to your sample		* Move the stage with the joystick to put the objective face to your sample
	* Move up the objective until oil touches the slides		* Move up the objective until oil touches the slides
	* Move up the stage with micrometric "screw" until you see your cells, or something moving		* Move up the stage with micrometric "screw" until you see your cells, or something moving
		+
	[[Paris|<<home]]		[[Paris|<<home]]

---

Revision as of 09:25, 13 July 2007

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 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) 6 Fluorescent single cells visualisation 6.1 Slide preparation 6.2 Turning on the microscope 6.3 Visualisation

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

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 can put wax around

Turning on the microscope

• turn on : the light, the microscope, the shutter, and the other thing
• then, launch metamorph
•  !! take care : not keeping the light on if we doesn't use it
•  !! do not switch on short after
• Stage to home : put down the objectives until max (load position)
• Put the proper stage on the microscope
• wait 5 min (the microscope is checking the ranges of movement
• (F2 : 50% of power)

Visualisation

• 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
• Move the stage with the joystick to put the objective face to your sample
• Move up the objective until oil touches the slides
• Move up the stage with micrometric "screw" until you see your cells, or something moving

<<home