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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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| - | This is an alternative to | + | =Agar Trap= |
| + | This is an alternative to [[Team:Cambridge-JIC/Guide/Transformation/Cocultivation| 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.] | ||
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| + | ==Protocol== | ||
| + | 1. [[Team:Cambridge-JIC/Guide/Care/PreparingSpores| Sterilise spores]] and plate them on [[Team:Cambridge-JIC/Guide/Materials/Media | 1/2 B5 + 1.2% agar plates]] on the same day (day 1) that you [[Team:Cambridge-JIC/Guide/Transformation/TransformingAgro|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 [[Team:Cambridge-JIC/Guide/Transformation/TransformingAgrobacteria| transformed agrobacteria]] in 1ml [[Team:Cambridge-JIC/Guide/Materials/Media| 1/2 GB + 100uM acetosyringone]] | ||
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3. Spread the agrobacteria suspension onto the spores, and leave for 30s-1min | 3. Spread the agrobacteria suspension onto the spores, and leave for 30s-1min | ||
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4. Using a pipette, extract any excess liquid. | 4. Using a pipette, extract any excess liquid. | ||
| - | 5. Leave under full illumination for approximately 3 days at | + | |
| + | 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. | 6. Scrape spores into a centrifuge tube containing 20ml of sterile water with 100ug/ml cefotaxime. | ||
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7. Using a 40um sterile cell-strainer, wash the spores with 150ml 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. | + | |
| + | 8. Plate spores on [[Team:Cambridge-JIC/Guide/Materials/Media| selective plates]], seal with micropore tape, and leave to grow. | ||
| + | |||
| + | ==Notes== | ||
| + | This method has the advantage of not requiring a full temperature shaker, and a ventilated illumination box can be hacked together at home. | ||
Revision as of 00:09, 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 1/2 GB + 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 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 full temperature shaker, and a ventilated illumination box can be hacked together at home.
