Mesoplasma florum:Electroporation

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