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Team:Cambridge-JIC/Guide/Care/PreparingSpores
From 2014.igem.org
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| - | Contamination (fungal in particular) is significant problem when cultivating and transforming marchantia. The best way to prevent it is by performing surface sterilisation of spores prior to germination, and then maintaining meticulous sterile technique for the remainder of the transformation steps. | + | Contamination (fungal in particular) is significant problem when cultivating and transforming marchantia. The best way to prevent it is by performing surface sterilisation of spores prior to germination, and then '''maintaining meticulous sterile technique for the remainder of the transformation steps.''' |
1. Prepare a gentle sterilising solution. A baby bottle sterilising solution is appropriately powerful. In the UK, 'Milton mini tablets' are widely available, and 1 tablet per 25ml is sufficient. | 1. Prepare a gentle sterilising solution. A baby bottle sterilising solution is appropriately powerful. In the UK, 'Milton mini tablets' are widely available, and 1 tablet per 25ml is sufficient. | ||
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2. Crush sporangia (spore heads) in a sterile 50ml centrifuge tube. Use approximately 1/2-1 sporangia's worth of spores per transformation. You can use a micropestle or a 15ml centrifuge tube. | 2. Crush sporangia (spore heads) in a sterile 50ml centrifuge tube. Use approximately 1/2-1 sporangia's worth of spores per transformation. You can use a micropestle or a 15ml centrifuge tube. | ||
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3. After vigorous crushing, add 1ml/sporangia of sterile water. Vortex thoroughly, and crush more. | 3. After vigorous crushing, add 1ml/sporangia of sterile water. Vortex thoroughly, and crush more. | ||
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4. When you are fed up of crushing, using a 40um cell-strainer, strain the contents of the centrifuge tube into another centrifuge tube. Spores are <40um in size, so pass through, but gunk does not. | 4. When you are fed up of crushing, using a 40um cell-strainer, strain the contents of the centrifuge tube into another centrifuge tube. Spores are <40um in size, so pass through, but gunk does not. | ||
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5. Aliquot into sterile microcentrifuge tubes (1ml per tube) | 5. Aliquot into sterile microcentrifuge tubes (1ml per tube) | ||
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6. Spin at 13000rpm for 1 minute, and carefully discard supernatant. | 6. Spin at 13000rpm for 1 minute, and carefully discard supernatant. | ||
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7. Resuspend in 1ml of sterilising solution. Leave for the recommended time (for Milton's mini, 20 minutes is sufficient) | 7. Resuspend in 1ml of sterilising solution. Leave for the recommended time (for Milton's mini, 20 minutes is sufficient) | ||
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8. Spin at 13000rpm for 1 minute, and carefully discard supernatant. | 8. Spin at 13000rpm for 1 minute, and carefully discard supernatant. | ||
| - | 9. Resuspend in 100ul sterile water, and plate on [[1/2 B5 + 1.2% agar plates]] | + | |
| - | 10. Leave upside down under full illumination for the required time (3 days for [[ | + | 9. Resuspend in 100ul sterile water, and plate on [[Team:Cambridge-JIC/Guide/Materials/Media| 1/2 B5 + 1.2% agar plates]] |
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| + | 10. Leave upside down under full illumination for the required time (3 days for [[Team:Cambridge-JIC/Guide/Materials/Transformation/AgarTrap]] and 5-7 days for [[Team:Cambridge-JIC/Guide/Transformation/Cocultivation]] | ||
Revision as of 00:48, 17 October 2014
Contamination (fungal in particular) is significant problem when cultivating and transforming marchantia. The best way to prevent it is by performing surface sterilisation of spores prior to germination, and then maintaining meticulous sterile technique for the remainder of the transformation steps.
1. Prepare a gentle sterilising solution. A baby bottle sterilising solution is appropriately powerful. In the UK, 'Milton mini tablets' are widely available, and 1 tablet per 25ml is sufficient.
2. Crush sporangia (spore heads) in a sterile 50ml centrifuge tube. Use approximately 1/2-1 sporangia's worth of spores per transformation. You can use a micropestle or a 15ml centrifuge tube.
3. After vigorous crushing, add 1ml/sporangia of sterile water. Vortex thoroughly, and crush more.
4. When you are fed up of crushing, using a 40um cell-strainer, strain the contents of the centrifuge tube into another centrifuge tube. Spores are <40um in size, so pass through, but gunk does not.
5. Aliquot into sterile microcentrifuge tubes (1ml per tube)
6. Spin at 13000rpm for 1 minute, and carefully discard supernatant.
7. Resuspend in 1ml of sterilising solution. Leave for the recommended time (for Milton's mini, 20 minutes is sufficient)
8. Spin at 13000rpm for 1 minute, and carefully discard supernatant.
9. Resuspend in 100ul sterile water, and plate on 1/2 B5 + 1.2% agar plates
10. Leave upside down under full illumination for the required time (3 days for Team:Cambridge-JIC/Guide/Materials/Transformation/AgarTrap and 5-7 days for Team:Cambridge-JIC/Guide/Transformation/Cocultivation
