Mesoplasma florum:Electroporation
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(Difference between revisions)
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* spin down again, remove supernatent | * spin down again, remove supernatent | ||
* resuspend the pellet in the remaining liquid | * resuspend the pellet in the remaining liquid | ||
| - | * add | + | * add 10 ml of chilled electroporation buffer |
| + | * spin down again, remove supernatent | ||
| + | * resuspend the pellet in the remaining liquid, bring the total volume to 200 μl | ||
* hold the cells on ice | * hold the cells on ice | ||
| - | + | * Add 4 μl of transposome or plasmid DNA for transformation | |
| - | * Add | + | * Mix and transfer to chilled 1 mm gap electroporation cuvettes |
| - | * Mix and transfer to | + | * Pulse the cuvette at 1.1 to 1.4 KV |
| - | * Pulse the cuvette at 1. | + | * Add 200 μl of chilled 1161 medium from a 10 ml tube, mix with the pipet |
| - | * Add 200 μl of chilled 1161 medium from a | + | * Transfer the cuvette contents back into the same tube, repeat |
| - | * Transfer the cuvette contents back into the same tube | + | * Place the 10 ml tubes into the 30° incubator for outgrowth for 2-3 hours |
| - | * Place the | + | |
** cells can be held at 4° following outgrowth | ** cells can be held at 4° following outgrowth | ||
| + | * spin down cells, discard supernatent | ||
| + | * resuspend in the remaining liquid, bring to 500 μl | ||
* Plate 100 μl aliquots on selective plates | * Plate 100 μl aliquots on selective plates | ||
| - | |||
* grow plates and medium at 30° for colonies or color change | * grow plates and medium at 30° for colonies or color change | ||
Revision as of 13:55, 25 September 2007
Transformation of Mesoplasma florum by electroporation
Materials
- overnight culture of Mesoplasma florum in ATCC 1161 medium
- chilled mycoplasma electroporation buffer
- 8 mM HEPES pH 7.4
- 272 mM sucrose
- chilled 1 mm electroporation cuvettes
- chilled ATCC 1161 medium aliquots of 1 ml in 2 ml tubes
- Selective medium or plates
- Tetracycline resistance from TetM is higher than 200 μg/ml
- Untransformed cells grow poorly at 4 μg/ml
- Selective plates are at 10 μg/ml
Protocol
- chill the centrifuge to 4°
- spin down 10 ml of overnight culture at 8000g for 5 minutes, remove the supernatent
- resuspend the pellet in the remaining liquid by vigorous vortexing
- add 10 ml of chilled electroporation buffer and mix
- spin down again, remove supernatent
- resuspend the pellet in the remaining liquid
- add 10 ml of chilled electroporation buffer
- spin down again, remove supernatent
- resuspend the pellet in the remaining liquid, bring the total volume to 200 μl
- hold the cells on ice
- Add 4 μl of transposome or plasmid DNA for transformation
- Mix and transfer to chilled 1 mm gap electroporation cuvettes
- Pulse the cuvette at 1.1 to 1.4 KV
- Add 200 μl of chilled 1161 medium from a 10 ml tube, mix with the pipet
- Transfer the cuvette contents back into the same tube, repeat
- Place the 10 ml tubes into the 30° incubator for outgrowth for 2-3 hours
- cells can be held at 4° following outgrowth
- spin down cells, discard supernatent
- resuspend in the remaining liquid, bring to 500 μl
- Plate 100 μl aliquots on selective plates
- grow plates and medium at 30° for colonies or color change
Notes 9/6/07: Arcing at 1.8 and 1.6 KV; possibly we need a second wash and/or some additional delay in the wash to remove more salt.
Gel loading tips do not work. Plating 125 ul seems to work well. Tested outgrowth at 2 ug/ml, 4 ug/ml, 8 ug/ml
