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Team:Imperial College London/Protocols Plant
From 2011.igem.org
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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 approx. 50 mg of <i>Arabidopsis</i> seeds in eppendorf tube (one tube per 250 ml Erlenmayer flask) <br> | + | - 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 approx. 4-5 minutes per tube (mix well) <br> | + | - Wash with 500 µl 70% EtOH for approx. 4-5 minutes per tube (mix well). <br> |
| - | - Remove 70% EtOH and replace with 500 µl of 50% bleach <br> | + | - Remove 70% EtOH and replace with 500 µl of 50% bleach. <br> |
| - | - Incubate for 20 min <br> | + | - Incubate for 20 min. <br> |
| - | - Wash 3x with sterile ddH2O to remove bleach<br> | + | - 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 approx. 200 rpm in constant light conditions<br> | + | - 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;"/> | ||
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<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> | <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 photo-oxidation of IAA.<br> | - 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 3 days<br> | + | - Growing is done at 23°C in darkness for 3 days.<br> |
- 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> | - 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 6 days</p> | + | - 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) were calculated for each replication by solving regression equations with y = y intercept + 2.</p> | - 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> | ||
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<h2>Making phytogels/ liquid media</h2> | <h2>Making phytogels/ liquid media</h2> | ||
| - | <p> | + | <p>Half-strength Murashige salt (2.1 g/L ddH2O) <br> |
| - | - Add 0.546 g MES salt (buffer) per liter of media <br> | + | - 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 10 g sucrose (normally from 1% solution).<br> |
| - | - Add 1% agarose = | + | - Add 1% agarose = 10 g/L if making phytogel.<br> |
| - | - Distribute into Erlenmayer flasks (125 ml/250 ml flask)<br> | + | - Distribute into Erlenmayer flasks (125 ml/250 ml flask).<br> |
| - | - Autoclave for at least 15 | + | - Autoclave for at least 15 min.</p> |
<h2>Seeding <i>Arabidopsis</i> into soil</h2><p> | <h2>Seeding <i>Arabidopsis</i> into soil</h2><p> | ||
| - | - Treat 12.5 kg of the soil with 1 | + | - Treat 12.5 kg of the soil with 1 L of 1 mM fungicide. <br> |
| - | - Rest the soil overnight <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> | + | - 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> | + | - Make a 1 cm depth hole into the soil.<br> |
| - | - Pipette each seed to the center of each hole <br> | + | - Pipette each seed to the center of each hole. <br> |
| - | - Cover the container with a plastic film to prevent water evaporation <br> | + | - Cover the container with a plastic film to prevent water evaporation. <br> |
| - | - Water the same amount of water everyday for 3 days | + | - Water with the same amount of water everyday for 3 days. After that water once every 2 days. |
</p> | </p> | ||
<hr style="color:#225323;"/> | <hr style="color:#225323;"/> | ||
Revision as of 16:15, 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 = 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
- Treat 12.5 kg of the 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 water everyday for 3 days. After that water once every 2 days.
