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From 2011.igem.org

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Revision as of 09:18, 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) - 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