Mesoplasma florum:Electroporation: Difference between revisions
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** 272 mM sucrose | ** 272 mM sucrose | ||
* chilled 1 mm electroporation cuvettes | * chilled 1 mm electroporation cuvettes | ||
* chilled ATCC 1161 medium aliquots of | * chilled ATCC 1161 medium aliquots of 10 ml in 15 ml centrifuge tubes | ||
* Selective medium or plates | * Selective medium or plates | ||
** Tetracycline resistance from TetM is higher than 200 μg/ml | ** Tetracycline resistance from TetM is higher than 200 μg/ml |
Revision as of 11:02, 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 10 ml in 15 ml centrifuge 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