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Team:Imperial College London/Protocols Plant
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- Autoclave for at least 15 minutes</p> | - Autoclave for at least 15 minutes</p> | ||
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| + | <h2>Seedling Arabidopsis in the soil</h2> | ||
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| + | - Treat 12.5 kg of the soil with 1 litre of 1 mM fungicide <br> | ||
| + | - Rest the soil overnight <br> | ||
| + | - Fill the soils in the container and water each 1 kg of soil in 100 ml of water <br> | ||
| + | - Make a 1 cm depth hole into the soil<br> | ||
| + | - Pipette each seed to the center of each hole <br> | ||
| + | - Cover the container with a plastic film to prevent water evaporation <br> | ||
| + | - Water the same amount of water everyday for 3 days and change to once in 2 days after 3 days time | ||
| + | </p> | ||
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Revision as of 12:32, 18 September 2011
Protocols
This page lists all the protocols used in our project. We have classified them into five main categories as follow.
Plant
Seeding
Vernalise seeds (this has to be done 2 to 3 days before seeding!)
- Weigh in appr. 50mg of arabidopsis seeds in eppendorf tube (one tube per 250 ml erlenmayer flask)
- Wash with 500 µl 70% EtOH for appr. 4-5 minutes per tube (mix well)
- Remove 70% EtOH and replace with 500µl 50% bleach
- Incubate for 20 minutes
- Wash several times with sterile ddH2O to remove bleach x3
- Vernalize seeds for 2-3 days
Some notes
- Growth conditions : flasks on a shaker at appr. 200 rpm at constant light conditions
- Grow seedlings for 5-6 days
Uptake
Overview : synthetic auxin is used to see the effect of Arabidopsis's 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, 10000uM indole-3-acetic acid (IAA).
- Medium preparation and seed sowing occurred under 0.5 pE m-2 sec-l incandescent light to minimize photooxidation of IAA.
- Growing is done at 23°C in darkness for three days
- After 3 days, hypocotyl and root lengths were measured on 10 plantslreplication. Data were normalized to lengths as a percentage of the control treatment and subjected to analysis of variance.
-Plants were transferred to light for a further six days
Some notes
- Concentrations of IAA causing 50% inhibition of root and hypocotyl growth (Isow) ere calculated for each replication by solving regression equations with y = y intercept + 2.
Making phytogels/liquid media
- Half strength Murashige salt (2.1g per liter ddH2O)
- Add 0.546g MES salt (buffer) per liter of media
- Adjust pH to 5.7-5.8 using 2M KOH
- add 10g sucrose (normally from 1% solution)
- Add 1% agarose = 10g/litre if making phytogel
- Distribute into erlenmayer flasks (125 ml/250ml flask)
- Autoclave for at least 15 minutes
Seedling Arabidopsis in the soil
- Treat 12.5 kg of the soil with 1 litre 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 the same amount of water everyday for 3 days and change to once in 2 days after 3 days time
