"
Team:Hannover/Protocols/Transformation/Arabidopsis
From 2014.igem.org
Protocols / Floral Dipping
Material: |
|
| Infiltration medium | 2.2 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 medium | 5 g/L Beef extract, 1 g/L Yeast extract, 5 g/L Peptone, 5 g/l Sucrose, 2 mM MgSO4 |
Protocol:
After E. coliCultivate 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. .
