Team:Toulouse/Notebook/Protocols

From 2014.igem.org

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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.
<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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<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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<b> Sap-like Medium (250 mL) (see references)</b> : <br>
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2,5g de tryptone<br>
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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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<br>
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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.
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Revision as of 23:53, 17 October 2014