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Team:Hannover/Protocols/Transformation/Arabidopsis
From 2014.igem.org
Protocols / Floral Dipping
Material: |
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| 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 | ||
| Plastic bags |
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.
Sterilisation of Seeds
Material: |
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| Sterile Whatman paper | |
| MS medium | 4.3 g MS basal salt, 0,5 g MES pH 5.7, 8 g Gelrite, 20 g sucrose, After autoclave, add MS vitamines |
Before transferring the ripened seeds on solid media, three steps of EtOH sterilization were performed.First, seeds were washed in 70 % EtOH, then washed in 96 % EtOH and finally kept for 10 min in 70 % EtOH again. Under aseptic conditions, seeds were dried on Whatman paper and equally distributed on herbicide containing, solid MS medium. Before placing the seeds in clima chambers, seeds were stored for 2 d at 4 °C.
