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Team:Imperial College London/Project/Arabidopsis/Protocols
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- Growth conditions : flasks on a shaker at appr. 200 rpm. Either under long day or constant light conditions<br> | - Growth conditions : flasks on a shaker at appr. 200 rpm. Either under long day or constant light conditions<br> | ||
- Grow seedlings for 5-6 days<br> | - Grow seedlings for 5-6 days<br> | ||
| + | </html> | ||
| + | |||
| + | <html> | ||
| + | |||
| + | '''Arabidopsis'''<br><br> | ||
| + | |||
| + | Seedling protocol | ||
| + | <br><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 (or 2x2) minutes per tube (mix well) <br> | ||
| + | |||
| + | |||
| + | - Remove 70% EtOH and replace with 500µl 50% bleach (alternatively 5% Calcium hypochloride) <br> | ||
| + | |||
| + | |||
| + | - Incubate for 20 minutes <br> | ||
| + | |||
| + | |||
| + | - Wash several times with sterile ddH2O to remove bleach x3 <br> | ||
| + | |||
| + | |||
| + | - Vernalize seeds for 2-3 days <br><br> | ||
| + | |||
| + | Prepare sterile medium<br><br> | ||
| + | |||
| + | |||
| + | - Half strength Murashige salt (2.1g per liter ddH2O) <br> | ||
| + | |||
| + | |||
| + | - Add 0.5g MES salt (buffer) per liter of media <br> | ||
| + | |||
| + | |||
| + | - Adjust pH to 5.7-5.8 using 2M KOH <br> | ||
| + | |||
| + | |||
| + | - If required add 10g sucrose (e.g. 1% solution) – yes <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 <br><br> | ||
| + | |||
| + | Some notes<br><br> | ||
| + | |||
| + | |||
| + | - Growth conditions : flasks on a shaker at appr. 200 rpm. Either under long day or constant light conditions <br> | ||
| + | |||
| + | |||
| + | - Grow seedlings for 5-6 days <br> | ||
</html> | </html> | ||
Revision as of 10:44, 22 July 2011
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)
- 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
'''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
