Mesoplasma florum:Electroporation
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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 100 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 0.5 ml of chilled electroporation buffer, mix
- hold the cells on ice
- Aliquot 50 μl of cells into chilled eppendorfs
- Add 1 μl of DNA for transformation
- Mix and transfer to a chilled 1 mm gap electroporation cuvette
- Pulse the cuvette at 1.75 KV twice, with a 5 second delay
- Add 200 μl of chilled 1161 medium from a 2 ml tube, mix with the pipet
- Transfer the cuvette contents back into the same tube
- transfer may be easier with a gel loading tip to reach into the gap
- Place the 2 ml tubes into the 30° incubator for outgrowth for 2-3 hours
- cells can be held at 4° following outgrowth
- Plate 100 μl aliquots on selective plates
- Transfer 100 μl aliquots into 10 ml of selective medium
- 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