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 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 1.2 KV
• 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
• 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