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Team:Cambridge-JIC/Guide/Transformation/Cocultivation
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=Cocultivation= | =Cocultivation= | ||
The time window for commencing cocultivation is when spores are 5-7 days old. | The time window for commencing cocultivation is when spores are 5-7 days old. | ||
| - | This protocol was first described in [http://pcp.oxfordjournals.org/content/49/7/1084 this paper] | + | This protocol was first described in [http://pcp.oxfordjournals.org/content/49/7/1084 this paper]. |
==Protocol== | ==Protocol== | ||
| - | [[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. | + | [[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. |
1. On day 3, make 10-15ml liquid cultures (LB) of agrobacteria from the [[Team:Cambridge-JIC/Guide/Transformation/TransformingAgro| transformation plates]], and leave for 18 hours to grow. It is recommended to use fresh plates for this step to ensure that the agro is nice and virulent. | 1. On day 3, make 10-15ml liquid cultures (LB) of agrobacteria from the [[Team:Cambridge-JIC/Guide/Transformation/TransformingAgro| transformation plates]], and leave for 18 hours to grow. It is recommended to use fresh plates for this step to ensure that the agro is nice and virulent. | ||
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2. When LB is saturated, spin cultures down at 3000g for 10 minutes. | 2. When LB is saturated, spin cultures down at 3000g for 10 minutes. | ||
| - | 3. Resuspend the pellet in 2ml [[Team:Cambridge-JIC/Guide/Materials/Media| 1/2 GB + 100uM acetosyringone]] | + | 3. Resuspend the pellet in 2ml [[Team:Cambridge-JIC/Guide/Materials/Media| 1/2 GB + 100uM acetosyringone + 5% sucrose]] |
4. Leave the resuspended culture shaking (120rpm is sufficient) at room temperature for 6 hours. Overnight is OK, but 6 hours is optimal. | 4. Leave the resuspended culture shaking (120rpm is sufficient) at room temperature for 6 hours. Overnight is OK, but 6 hours is optimal. | ||
| - | 5. Scrape spores into sterile conical flasks (autoclave them first) containing 25ml [[1/2 | + | 5. Scrape spores into sterile conical flasks (autoclave them first) containing 25ml [[Team:Cambridge-JIC/Guide/Materials/Media| 1/2 GB + 100uM acetosyringone + 5% sucrose]] |
6. Add 1ml of the agrobacterium mixture that has been shaking for 6 hours. | 6. Add 1ml of the agrobacterium mixture that has been shaking for 6 hours. | ||
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[http://pcp.oxfordjournals.org/content/49/7/1084 Agrobacterium-Mediated Transformation of the Haploid Liverwort Marchantia polymorpha L., an Emerging Model for Plant Biology, Ishizaki et al.] | [http://pcp.oxfordjournals.org/content/49/7/1084 Agrobacterium-Mediated Transformation of the Haploid Liverwort Marchantia polymorpha L., an Emerging Model for Plant Biology, Ishizaki et al.] | ||
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Latest revision as of 15:36, 17 October 2014
Cocultivation
The time window for commencing cocultivation is when spores are 5-7 days old.
This protocol was first described in [http://pcp.oxfordjournals.org/content/49/7/1084 this paper].
Protocol
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.
1. On day 3, make 10-15ml liquid cultures (LB) of agrobacteria from the transformation plates, and leave for 18 hours to grow. It is recommended to use fresh plates for this step to ensure that the agro is nice and virulent.
2. When LB is saturated, spin cultures down at 3000g for 10 minutes.
3. Resuspend the pellet in 2ml 1/2 GB + 100uM acetosyringone + 5% sucrose
4. Leave the resuspended culture shaking (120rpm is sufficient) at room temperature for 6 hours. Overnight is OK, but 6 hours is optimal.
5. Scrape spores into sterile conical flasks (autoclave them first) containing 25ml 1/2 GB + 100uM acetosyringone + 5% sucrose
6. Add 1ml of the agrobacterium mixture that has been shaking for 6 hours.
7. Shake flasks (120rpm is sufficient) under full illumination for between 36-72 hours.
8. Using a 40um sterile filter, wash the spores with 150ml of sterile water with 100ug/ml cefotaxime.
9. Plate spores on selective plates.
Notes
Optimal transformation efficiency is observed when spores are 7 days old. For a detailed study of optimal conditions, see the source paper:
[http://pcp.oxfordjournals.org/content/49/7/1084 Agrobacterium-Mediated Transformation of the Haploid Liverwort Marchantia polymorpha L., an Emerging Model for Plant Biology, Ishizaki et al.]
