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Revision as of 15:15, 17 October 2014

Agar Trap

This is an alternative to cocultivation method step, and is from: [http://pcp.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=24259681 AgarTrap: a simplified Agrobacterium-mediated transformation method for sporelings of the liverwort Marchantia polymorpha L., Tsuboyama S. and Kodama Y.]

Protocol

1. Sterilise spores and plate them on 1/2 B5 + 1.2% agar plates on the same day (day 1) that you transform agrobacteria. Leave them to germinate under full illumination. Using an average of 0.5-1 sporehead's worth of spores gives a good quantity of transformants.

2. When spores are 3 days old, suspend a colony of transformed agrobacteria in 1ml MMA buffer

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 room temperature, with ventilation, 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, seal with micropore tape, and leave to grow.

Notes

This method has the advantage of not requiring a room temperature shaker under full illumination, and a ventilated illumination box can be hacked together at home.