Team:Toulouse/Notebook/Protocols

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<i>NB: for kit plate, resuspend the well in 10 µL of sterile water</i>
<i>NB: for kit plate, resuspend the well in 10 µL of sterile water</i>
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- Put the tubes 20 minutes in the ice
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- Put the tubes 20 minutes in the ice </i>
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- Put the tubes 2 minutes at 42°C in the water bath
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- Put the tubes 2 minutes at 42°C in the water bath to create the thermic shock
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- Put the tubes back in ice immediately to create the thermic shock
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- Put the tubes back in ice immediately  
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- Add 1mL of LB medium
- Add 1mL of LB medium
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<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200 µL of buffer 1 is added to resuspend the pellet, 400 µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300 µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.
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<br/><i>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200 µL of buffer 1 is added to resuspend the pellet, 400 µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300 µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.
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600 µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100 µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse.  
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600 µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100 µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse. </i>
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<br> <b>Buffer 1:</b> Tris 10 mM pH 8 + EDTA 1mM
<br> <b>Buffer 1:</b> Tris 10 mM pH 8 + EDTA 1mM
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</I></p>
</I></p>
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<p class="title1" id="select4"> Cloning </p>
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<p class="title1" id="select4">Cloning </p>
<p class="texte">Cloning is the step after taking the competent cells, transforming the BioBricks and miniprep them.
<p class="texte">Cloning is the step after taking the competent cells, transforming the BioBricks and miniprep them.
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<p class="texte">
<p class="texte">
<B> Day 0 </B>
<B> Day 0 </B>
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<br/>- Streak out the <i>Bacillus</i> strain and plate this on an LB agar plate overnight at 37°C</p>
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<br/>- Streak out the <i>B. subtilis</i> strain and plate this on an LB agar plate overnight at 37°C</p>
<p class="texte"><B> Day 1 </B>
<p class="texte"><B> Day 1 </B>
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<br/>- Pick up a nice big colony of <I>B. Subtilis </I> strain and drop it in 2ml of completed 1x MC
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<br/>- Pick up a nice big colony of <I>B. subtilis </I> strain and drop it in 2ml of completed 1x MC
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<br/>
- Grow at 37°C for 5 hours
- Grow at 37°C for 5 hours
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<p class="title1" id="select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</p>
<p class="title1" id="select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</p>
<p class="texte">
<p class="texte">
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<br>- Plate the transformed <i>Bacillus</i> strain on a selective medium (LB + spectinomycin) overnight  
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<br>- Plate the transformed <i>B. subtilis</i> strain on a selective medium (LB + spectinomycin) overnight  
<br>- The obtained clones are then plated on different media:  Medium Competence (Thr+), Medium Competence (Thr-) and LB + Spectinomycin.  
<br>- The obtained clones are then plated on different media:  Medium Competence (Thr+), Medium Competence (Thr-) and LB + Spectinomycin.  
<br>When the plasmid is integrated, the clone can grow on minimum medium with threonine and on LB + Spectinomycin but can not grow on the minimum medium without thronine.
<br>When the plasmid is integrated, the clone can grow on minimum medium with threonine and on LB + Spectinomycin but can not grow on the minimum medium without thronine.
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<p class="texte">
<p class="texte">
<br>- Put 200 µl of the different chemoattractants in the wells of the ELISA plate and pipet 15 µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). The volume in the tips must be marked.<br>
<br>- Put 200 µl of the different chemoattractants in the wells of the ELISA plate and pipet 15 µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). The volume in the tips must be marked.<br>
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<br><i>NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on <i>Ceratocystis platani</i> wall.<br>  
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<br><i>NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on <i>Ceratocystis platani</i> wall.</i><br>
<br>- Put the tips with chemoattractants in 300 µL of the bacterial solution in exponential growth phase in the ELISA plate.
<br>- Put the tips with chemoattractants in 300 µL of the bacterial solution in exponential growth phase in the ELISA plate.
<br>- Let the installation settle for 1 hour at room temperature.
<br>- Let the installation settle for 1 hour at room temperature.
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<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild type strains or copper sulfate at 10 and 20mg/mL.
<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild type strains or copper sulfate at 10 and 20mg/mL.
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</p>
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<p class= "texte">
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<b> Sap-like Medium (250 mL) (see references)</b>: <br>
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2,5g de tryptone
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<br>1,25g de YE
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<br>2,5g de NaCl
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<br>Glucose : 1,175g
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<br>Fructose : 1,125g
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<br>Sucrose : 0,125g
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<br>Inositol : 0,084 g
<p class="title2">Fungicide test: <i>in planta</i> assay</p>
<p class="title2">Fungicide test: <i>in planta</i> assay</p>
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The next step begins with the preparation of the fungal samples. Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and  through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until it reaches an OD of 2.5 at 600nm. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf, 5µl of the fungal suspension is deposited (using beveled tips because it is too viscous). As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.
The next step begins with the preparation of the fungal samples. Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and  through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until it reaches an OD of 2.5 at 600nm. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf, 5µl of the fungal suspension is deposited (using beveled tips because it is too viscous). As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.
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</p>
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<p class="texte">
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<b>References</b><br>
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Véronique Amiard, Annette Morvan-Bertrand, Jean-Bernard Cliquet, Jean-Pierre Billard,
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Claude Huault, Jonas P. Sandström, and Marie-Pascale Prud’homme. <b>Carbohydrate and amino
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acid composition in phloem sap of Lolium perenne L. before and after defoliation</b>. Can. J. Bot.
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Vol. 82: 1594–1601, 2004.</p>
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Latest revision as of 03:37, 18 October 2014