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 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

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