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Team:Imperial College London/Protocols Plant
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<p><b>Vernalise seeds (this has to be done 2 to 3 days before seeding!)</b><br> | <p><b>Vernalise seeds (this has to be done 2 to 3 days before seeding!)</b><br> | ||
| - | - Weigh | + | - Weigh approx. 50 mg of <i>Arabidopsis</i> seeds in eppendorf tube (one tube per 250 ml Erlenmayer flask) <br> |
| - | - Wash with 500 µl 70% EtOH for | + | - Wash with 500 µl 70% EtOH for approx. 4-5 minutes per tube (mix well) <br> |
| - | - Remove 70% EtOH and replace with | + | - Remove 70% EtOH and replace with 500 µl of 50% bleach <br> |
| - | - Incubate for 20 | + | - Incubate for 20 min <br> |
| - | - Wash | + | - Wash 3x with sterile ddH2O to remove bleach<br> |
- Vernalize seeds for 2-3 days</p> | - Vernalize seeds for 2-3 days</p> | ||
<p><b>Some notes</b><br> | <p><b>Some notes</b><br> | ||
| - | - Growth conditions : flasks on a shaker at | + | - Growth conditions : flasks on a shaker at approx. 200 rpm in constant light conditions<br> |
- Grow seedlings for 5-6 days</p> | - Grow seedlings for 5-6 days</p> | ||
<hr style="color:#225323;"/> | <hr style="color:#225323;"/> | ||
| Line 22: | Line 22: | ||
<h2>Uptake</h2> | <h2>Uptake</h2> | ||
| - | <p><b>Overview : synthetic auxin is used to see the effect | + | <p><b>Overview : synthetic auxin is used to see the effect on <i>Arabidopsis</i> root growth. Variation in auxin concentrations is applied to see the sensitivity of auxin in <i>Arabidopsis</i>.</b></p> |
| - | <p>- 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, | + | <p>- To test auxin-sensitivity, <i>Arabidopsis</i> 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). <br> |
| - | - Medium preparation and seed sowing occurred under 0.5 pE m-2 sec-l incandescent light to minimize | + | - Medium preparation and seed sowing occurred under 0.5 pE m-2 sec-l incandescent light to minimize photo-oxidation of IAA.<br> |
| - | - Growing is done at 23°C in darkness for | + | - Growing is done at 23°C in darkness for 3 days<br> |
| - | - After 3 days, hypocotyl and root lengths were measured on 10 | + | - 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. <br> |
| - | -Plants were transferred to light for a further | + | - Plants were transferred to light for a further 6 days</p> |
<p><b>Some notes</b><br> | <p><b>Some notes</b><br> | ||
| - | - Concentrations of IAA causing 50% inhibition of root and hypocotyl growth (Isow) | + | - 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.</p> |
<hr style="color:#225323;"/> | <hr style="color:#225323;"/> | ||
| - | <h2>Making phytogels/liquid media</h2> | + | <h2>Making phytogels/ liquid media</h2> |
| - | <p>- Half strength Murashige salt (2. | + | <p>- Half-strength Murashige salt (2.1 g/L ddH2O) <br> |
| - | - Add 0. | + | - Add 0.546 g MES salt (buffer) per liter of media <br> |
- Adjust pH to 5.7-5.8 using 2M KOH <br> | - Adjust pH to 5.7-5.8 using 2M KOH <br> | ||
| - | - | + | - Add 10 g sucrose (normally from 1% solution)<br> |
- Add 1% agarose = 10g/litre if making phytogel<br> | - Add 1% agarose = 10g/litre if making phytogel<br> | ||
| - | - Distribute into | + | - Distribute into Erlenmayer flasks (125 ml/250 ml flask)<br> |
- Autoclave for at least 15 minutes</p> | - Autoclave for at least 15 minutes</p> | ||
| - | <h2> | + | <h2>Seeding <i>Arabidopsis</i> into soil</h2><p> |
- Treat 12.5 kg of the soil with 1 litre of 1 mM fungicide <br> | - Treat 12.5 kg of the soil with 1 litre of 1 mM fungicide <br> | ||
Revision as of 16:07, 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 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
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.
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 = 10g/litre if making phytogel
- Distribute into Erlenmayer flasks (125 ml/250 ml flask)
- Autoclave for at least 15 minutes
Seeding Arabidopsis into 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
