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Team:Imperial College London/Experiments
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'''Arabidopsis'''<br><br> | '''Arabidopsis'''<br><br> | ||
| - | + | Seedling protocol | |
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- Vernalize seeds for 2-3 days <br><br> | - Vernalize seeds for 2-3 days <br><br> | ||
| - | + | Prepare sterile medium<br><br> | |
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- Autoclave for at least 15 minutes <br><br> | - Autoclave for at least 15 minutes <br><br> | ||
| - | + | Some notes<br><br> | |
Latest revision as of 22:40, 21 July 2011
'''Arabidopsis'''
Seedling protocol
- 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 (or 2x2) minutes per tube (mix well)
- Remove 70% EtOH and replace with 500µl 50% bleach (alternatively 5% Calcium hypochloride)
- 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 0.5g MES salt (buffer) per liter of media
- Adjust pH to 5.7-5.8 using 2M KOH
- If required add 10g sucrose (e.g. 1% solution) – yes
- 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. Either under long day or constant light conditions
- Grow seedlings for 5-6 days
