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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> | ||
| + | - Weigh in appr. 50mg of arabidopsis seeds in eppendorf tube (one tube per 250 ml erlenmayer flask) <br> | ||
| + | - Wash with 500 µl 70% EtOH for appr. 4-5 minutes per tube (mix well) <br> | ||
| + | - Remove 70% EtOH and replace with 500µl 50% bleach <br> | ||
| + | - Incubate for 20 minutes <br> | ||
| + | - Wash several times with sterile ddH2O to remove bleach x3 <br> | ||
| + | - Vernalize seeds for 2-3 days</p> | ||
| + | <p><b>Prepare sterile medium</b><br> | ||
| + | - Half strength Murashige salt (2.1g per liter ddH2O) <br> | ||
| + | - Add 0.546g MES salt (buffer) per liter of media <br> | ||
| + | - Adjust pH to 5.7-5.8 using 2M KOH <br> | ||
| + | - add 10g sucrose (normally from 1% solution)<br> | ||
| + | - Add 1% agarose = 10g/litre if making phytogel<br> | ||
| + | - Distribute into erlenmayer flasks (125 ml/250ml flask)<br> | ||
| + | - Autoclave for at least 15 minutes</p> | ||
| + | <p><b>Some notes</b><br> | ||
| + | - Growth conditions : flasks on a shaker at appr. 200 rpm at constant light conditions<br> | ||
| + | - Grow seedlings for 5-6 days</p> | ||
<h2>Uptake</h2> | <h2>Uptake</h2> | ||
<hr style="color:#225323;"/> | <hr style="color:#225323;"/> | ||
Revision as of 18:09, 16 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
Prepare sterile medium
- 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
Some notes
- Growth conditions : flasks on a shaker at appr. 200 rpm at constant light conditions
- Grow seedlings for 5-6 days
