Team:Imperial College London/Project/Arabidopsis/Protocols

From 2011.igem.org

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(Created page with "'''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) ...")
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'''Seedling protocol'''
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<html>
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<h1>Seedling protocol</h1>
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- Weigh in appr. 50mg of arabidopsis seeds in eppendorf tube (one tube per 250 ml erlenmayer flask)  
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<br><br>
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- Wash with 500 µl 70% EtOH for appr. 4-5 (or 2x2) minutes per tube (mix well)  
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- Weigh in appr. 50mg of arabidopsis seeds in eppendorf tube (one tube per 250 ml erlenmayer flask) <br>
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- Remove 70% EtOH and replace with 500µl 50% bleach (alternatively 5% Calcium hypochloride)  
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- Wash with 500 µl 70% EtOH for appr. 4-5 (or 2x2) minutes per tube (mix well) <br>
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- Incubate for 20 minutes  
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- Remove 70% EtOH and replace with 500µl 50% bleach (alternatively 5% Calcium hypochloride) <br>
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- Wash several times with sterile ddH2O to remove bleach x3  
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- Incubate for 20 minutes <br>
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- Wash several times with sterile ddH2O to remove bleach x3 <br>
- Vernalize seeds for 2-3 days  
- Vernalize seeds for 2-3 days  
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<br><br>
Prepare sterile medium
Prepare sterile medium
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<br><br>
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- Half strength Murashige salt (2.1g per liter ddH2O) <br><br
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- Add 0.5g MES salt (buffer) per liter of media <br>
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- Adjust pH to 5.7-5.8 using 2M KOH <br>
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- If required add 10g sucrose (e.g. 1% solution) – yes <br>
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- Add 1% agarose = 10g/litre if making phytogel<br>
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- Distribute into erlenmayer flasks (125 ml/250ml flask) <br>
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- Autoclave for at least 15 minutes <br>
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<br>
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Some notes<br><br>
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- Half strength Murashige salt (2.1g per liter ddH2O)
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- Growth conditions : flasks on a shaker at appr. 200 rpm. Either under long day or constant light conditions<br>
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- Add 0.5g MES salt (buffer) per liter of media
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- Grow seedlings for 5-6 days<br>
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- Adjust pH to 5.7-5.8 using 2M KOH
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</html>
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- 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
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-
 
+
-
- Growth conditions : flasks on a shaker at appr. 200 rpm. Either under long day or constant light conditions  
+
-
- Grow seedlings for 5-6 days
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Revision as of 09:20, 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

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