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From 2011.igem.org

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Protocols

This page lists all the protocols used in our project. We have classified them into five main categories as follow.

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Plant

Seeding

Vernalise seeds (this has to be done 2 to 3 days before seeding!)
- Weigh approx. 50 mg of Arabidopsis seeds in eppendorf tube (one tube per 250 ml Erlenmayer flask).
- Wash with 500 µl 70% EtOH for approx. 4-5 minutes per tube (mix well).
- Remove 70% EtOH and replace with 500 µl of 50% bleach.
- Incubate for 20 min.
- Wash 3x with sterile ddH2O to remove bleach.
- Vernalize seeds for 2-3 days.

Some notes
- Growth conditions : flasks on a shaker at approx. 200 rpm in constant light conditions.
- Grow seedlings for 5-6 days.

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Uptake

Overview : synthetic auxin is used to see the effect on Arabidopsis root growth. Variation in auxin concentrations is applied to see the sensitivity of auxin in Arabidopsis.

- To test auxin-sensitivity, Arabidopsis seeds were sown onto medium as given above and supplemented with 0, 0.00001, 0.0001, 0.01, 1, 100, 10000 µM indole-3-acetic acid (IAA).
- Medium preparation and seed sowing occurred under 0.5 pE m-2 sec-l incandescent light to minimize photo-oxidation of IAA.
- Growing is done at 23°C in darkness for 3 days.
- After 3 days, hypocotyl and root lengths were measured on 10 replicate plants. Data were normalized to length as a percentage of the control treatment and subjected to analysis of variance.
- Plants were transferred to light for a further 6 days.

Some notes
- Concentrations of IAA causing 50% inhibition of root and hypocotyl growth (Isow) were calculated for each replication by solving regression equations with y = y intercept + 2.

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Making phytogels/ liquid media

Half-strength Murashige salt (2.1 g/L ddH2O)
- Add 0.546 g MES salt (buffer) per liter of media.
- Adjust pH to 5.7-5.8 using 2M KOH.
- Add 10 g sucrose (normally from 1% solution).
- Add 1% agarose = 10 g/L if making phytogel.
- Distribute into Erlenmayer flasks (125 ml/250 ml flask).
- Autoclave for at least 15 min.

Seeding Arabidopsis into soil in soil erosion experiment

- set up the experiment with 18 (3 columns at 10 cm side x 6 rows on 20 cm side) where Arabidopsis are seeded in 3 rectangular pots which represented each concentration of IAA. Together with 2 controls (one where no IAA was watered and one where no Arabidopsis was seeded), 30 pots in total.
- Treat 12.5 kg of the M2 soil with 1 L of 1 mM fungicide.
- Rest the soil overnight.
- Fill the soils in the container and water each 1 kg of soil in 100 ml of water.
- Make a 1 cm depth hole into the soil.
- Pipette each seed to the center of each hole.
- Cover the container with a plastic film to prevent water evaporation.
- Water with the same amount of IAA concentrations everyday for 3 days. After that water once every 2 days.

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Obtaining the water mass retained in soil erosion experiment

- At 10 minutes after final watering, 10 cm³ of soil at a region between 4 plant roots is collected for measuring wet mass.
- The soil is incubated for 1 day to get rid of water to achieve dry mass. The difference between the wet and the dry mass was the water hold up in the soil.
- The plants are left 2 days with no watering and the same procedure to measure water hold up was repeated. The smaller the difference of water retained between 10 min and 2 days after watering, the more water was retained by the soil

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