Mesoplasma florum:Electroporation

Transformation of Mesoplasma florum by electroporation

Materials

 * overnight culture of Mesoplasma florum in ATCC 1161 medium
 * preparing 10 ml cultures with 1, 10, 100, and 1000 &mu;l of infected cultures assures that one of these will be ready the next day at the correct OD level. Select cultures which are just changing color.
 * chilled mycoplasma electroporation buffer
 * 8 mM HEPES pH 7.4
 * 272 mM sucrose (93.1 g/l)
 * chilled 1 mm electroporation cuvettes
 * 10 ml chilled ATCC 1161 medium
 * Selective medium or plates
 * Tetracycline resistance from TetM is higher than 200 &mu;g/ml
 * Untransformed cells grow poorly at 4 &mu;g/ml
 * Tet selective plates are at 15 &mu;g/ml

Protocol

 * chill the centrifuge to 4&deg;
 * 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 1200 &mu;l with EPB
 * Freeze 400 &mu;l aliquots at -80 indefinitely or use immediately
 * Add 16 &mu;l of transposome or plasmid DNA for transformation
 * Mix and transfer to four chilled 1 mm gap electroporation cuvettes
 * Pulse the cuvette at 1.5 KV
 * Add ml of chilled 1161 medium immediately, mix with the pipet, cover
 * Place the cuvettes into the 30&deg; incubator for outgrowth for 50 minutes
 * cells can be held at 4&deg; following outgrowth
 * dilute 1 &mu;l of the culture into 1 ml of 1161 medium, vortex, and dilute again into 1 ml. Plate 200 &mu;l on a nonselective 1161 plate for counting untransformed CFUs.
 * Plate 360 &mu;l aliquots on three selective plates
 * grow plates for 1.5 - 2 days at 30&deg; for colonies