Team:Hannover/Protocols/Transformation/Arabidopsis

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<p><h4>Protocol:</h4></p>
<p><h4>Protocol:</h4></p>
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<p class="text">After <i>E. coli</i>Cultivate <i>R. radiobacter</i> cells in 5 ml antbiotics containing YEB media for 2 days at 28 °C. To start your main culture, add 1 ml of that suspension to 50 ml of antibiotics containing YEB medium and incubate it for 1 d at 28 °C. When the cell culture reached an optical density (OD600) of ~1.0, the bacteria were placed on ice and centrifuged for 15 min at 3000 rpm and 4 °C. While the supernatant was decanted, the precipitate was dissolved in 200 ml infiltration medium. The Inflorescence (mind. 15 cm) of Arabidopsis plants were dipped in the solution for 6 min. To minimize contaminations, remove already opened flowers and seed containing siliques. Place the pot in a horizontal position on a tray and cover all plants in plastic bags. The bags can be removed at the next day. .</p></div>
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<p class="text">Cultivate <i>R. radiobacter</i> cells in 5 ml antbiotics containing YEB media for 2 days at 28 °C. To start your main culture, add 1 ml of that suspension to 50 ml of antibiotics containing YEB medium and incubate it for 1 d at 28 °C. When the cell culture reached an optical density (OD600) of ~1.0, the bacteria were placed on ice and centrifuged for 15 min at 3000 rpm and 4 °C. While the supernatant was decanted, the precipitate was dissolved in 200 ml infiltration medium. The Inflorescence (mind. 15 cm) of Arabidopsis plants were dipped in the solution for 6 min. To minimize contaminations, remove already opened flowers and seed containing siliques. Place the pot in a horizontal position on a tray and cover all plants in plastic bags <span id='a1'</span>. The bags can be removed at the next day. .</p></div>
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Revision as of 17:20, 15 October 2014

Protocols / Floral Dipping

Material:
Infiltration medium2.2 g/L 0.5x MS medium,
5% Saccharose,
0.5 g/L MES,
0.03% Silwett L-77, pH 5.7 (adjust with 2N NaOH)
YEB medium5 g/L Beef extract,
1 g/L Yeast extract,
5 g/L Peptone,
5 g/l Sucrose,
2 mM MgSO4

Protocol:

Cultivate R. radiobacter cells in 5 ml antbiotics containing YEB media for 2 days at 28 °C. To start your main culture, add 1 ml of that suspension to 50 ml of antibiotics containing YEB medium and incubate it for 1 d at 28 °C. When the cell culture reached an optical density (OD600) of ~1.0, the bacteria were placed on ice and centrifuged for 15 min at 3000 rpm and 4 °C. While the supernatant was decanted, the precipitate was dissolved in 200 ml infiltration medium. The Inflorescence (mind. 15 cm) of Arabidopsis plants were dipped in the solution for 6 min. To minimize contaminations, remove already opened flowers and seed containing siliques. Place the pot in a horizontal position on a tray and cover all plants in plastic bags . The bags can be removed at the next day. .

For R. radiobacter based plant transformation humidity is an important factor. The more humid, the better is the infection.





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