Difference between revisions of "Team:Toulouse/Experiments"

To run tests with varroas it is necessary to get them back from beehive directly because they cannot live without bees.

In the begining we tested high and low concentrations and in function of results we adapted concentrations. In the end we worked with these concentrations:

• Butyric acid : 218 mM, 109 mM, 10.9 mM, 5.45 mM and 1.09 mM
• Formic acid : 100 mM, 10 mM, 1 mM 500 µM, 100 µM, 50 µM and 25 µM

Note 1: We used culture in M9 because with the “Acids production tests” we had data on this medium.
Note 2: 10 mM NaHCO₃ solution was used because pH was 8.3 so it would permit acid gas solubilisation.

• 1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37 °C
• 2. Pick a single fresh colony to inoculate 5 mL of YETM medium. Grow over night at 37 °C.
• Do not vortex cells at any time after this point in the procedure
• 3. Dilute 1 mL of culture into 50 mL YETM medium prewarmed to 37 °C
• Grow at 37 °C for 2 hours with agitation
• Volumes can be scaled up 5X and all of the 5 mL overnight culture can be used
• 4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes
• 5. Centrifuge for 10 minutes at 2,000 rpm at 4 °C. Immediately aspirate off all of the supernatant
• Do not allow cells to warm above 4 °C at any time in this procedure
• 6. Resuspend cells in 10 mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette
• 7. Incubate cells on ice for 5 minutes
• 8. Repeat step 5
• 9. Resuspend cells in 2 mL of ice-cold TFB2 with gentle re-pipetting. Use micropipet tip (plastic)
• 10. Incubate cells on ice for 15 minutes
• Cells may be used for transformation or frozen
• To freeze: aliquot cell in 200 μL volumes into prechilled 0.5 mL microfuge tube (on ice)
• Freeze immediately in liquid nitrogen
• Store cells frozen at -80 °C

• 1. If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed.
  Immedately place on ice for about 5 minutes
• 2. Add to 1,5 mL eppendorff on ice: 2-3 μL iGEM plate or 1 μL plasmid or 10 μL ligation.
• 3. Add 100 μL of competent cells and mix by gentle re-pipetting
• 4. Incubate cells on ice for 20-30 minutes
• 5. Heat shock the cells exactly 90 seconds at 42 °C
• 6. Return cells on ice for 2 minutes
• 7. Add 1 mL of YETM medium. Incubate at 37 °C for 45-60 minutes with slow gentle shaking
• 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate overnight at 37°C

• 1. Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)
• 2. Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic
• 3. Let the culture grow overnight at 37 °C in a shaking incubator
• 4. Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55 °C
• 5. Keep the tubes at -20 °C

• 1. Prepare a 1 % or 2 % electrophoresis agarose gel with 0.5 X TAE buffer
• 2. Put 20 µL of sample + 6 µL of marker (1 kb for 1 % gel and 100 pb for 2 %) into the well
• 3. Migration for 30 min at 100 V or 1 hour at 50 V
• 4. Bathe 10 minutes in BET
• 5. Wash in water for 5 minutes
• 6. The gel extraction is realized thanks to the QIAGEN Gel Extraction Kit


• Two ways to inactivate the enzymes for the vector
• Use of DNA Clean up kit for the DNA fragment above 200 pb
• Heat inactivation at 95 °C for 10 minutes

• 1a. Take 10 µL of the ligation mix for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol
• 1b. Positive control: take 10 µL of the ligation mix for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol
• 1c. First restriction enzyme digestion control: take 10 µL of the corresponding digestion mix (First step) for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol
• 1d. Second restriction enzyme digestion control: take 10 µL of the corresponding digestion mix (First step) for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol
• 1e. Negative control: take 10 µL of the corresponding mix for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol
• 2. Plate the solution on selective medium overnight at 37 °C

• 1. Design the tailed-oligonucleotides for the vector and the inserts. The tail of the 5’oligonucleotide must be the 20 last nucleotides of the previous fragment and the tail of the 3’oligonucleotide must be the 20 first nucleotides of the next fragment.
• 2. Amplify the different fragments with the previously designed oligonucleotides
• 3. Clean the PCR products either using a spin column purification kit or by digesting with DpnI. For NEB DpnI, mix the different PCR products together, add 10X CutSmart Buffer and DpnI (1 µL in a 20 µL mix); incubate at 37°C for 20 minutes and inactive at 80 °C during 30 minutes.
• 4. Set up the In Fusion cloning reaction :



	Ligation	Control
5X In-Fusion HD Enzyme Premix	2 µL	-
Clean PCR mix	4 µL	4 µL
MilliQ water nuclease free	4 µL	6 µL
Total	10 µL	10 µL



• 5. Incubate at 50 °C during 15 min and then cool on ice
• 6. Transform commercial ultra-competent cells (10⁸ to 10⁹ cfu/µg DNA) with 2,5 µL of the ligation using provided with the competent cells. Plate several 10-fold dilutions of the transformation mix.