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Team:Cambridge-JIC/Guide/Transformation/AgarTrap
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(Created page with "This is an alternative to the cocultivation step, and a full protocol is described in detail in ... 1. Sterilise spores and plate them on [[1/2 B5 + 1.2% agar plates...")
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Revision as of 01:17, 16 October 2014
This is an alternative to the cocultivation step, and a full protocol is described in detail in ...
1. Sterilise spores and plate them on 1/2 B5 1.2% agar plates and leave to germinate at full illumination 2. When spores are 3 days old, suspend a colony of transformed agrobacteria in 1ml 1/2 B5 + 100uM acetosyringone. 3. Spread the agrobacteria suspension onto the spores, and leave for 30s-1min 4. Using a pipette, extract any excess liquid. 5. Leave under full illumination for approximately 3 days at RT under full illumination 6. Scrape spores into a centrifuge tube containing 20ml of sterile water with 100ug/ml cefotaxime. 7. Using a 40um sterile cell-strainer, wash the spores with 150ml of sterile water with 100ug/ml cefotaxime. 8. Plate spores on selective plates, and leave to grow.
