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Advanced Preparation: -Prepare 25 mL of Mycoplasma spp. culture to MID-LOG PHASE -Chill a 0.1cm cuvette at -20°C -Prepare electroporation buffer (1 mL recipe below) fresh for each transformation, and warm to the organism’s optimal growth temperature -1M HEPES 8 μL -1M sucrose 272 μL -dH2O 720 μL ***All components must be kept sterile***

Performance of Transformation: 1.) Harvest cells by centrifugation at room temperature 2.) Resuspend cell pellet in 1 mL pre-warmed electroporation buffer and centrifuge for 20 minutes at room temperature 3.) Decant supernatant and repeat step 2. 4.) Decant supernatant and resuspend cell pellet in 60 μL electroporation buffer 5.) Add 20 μg transposon-bearing plasmid (e.g.pISM2062, pTF20, psPuro) DNA and mix gently 6.) Incubate on ice for 15 minutes. 7.) Transfer cell/DNA suspension to chilled cuvette and immediately pulse. a. Bio-Rad Gene Pulser II, set at 2.5 kV, 100Ω, 25 μF 8.) Add 1 mL cold SP-4 medium (or appropriate growth medium for transformed species) and mix by inversion 9.) Incubate cells at room temperature for 10 minutes. 10.Allow 1-2 or 2-3 hours (depending on growth rate of transformed species) at optimal growth temperature (30 or 37°C) for recovery 11.)Pipet 100 μL onto SP-4 plates containing 10 μg/mL tetracycline (for tetM selection) (or appropriate selective medium) a. Pipet 100 μL onto non-selective plates to ensure viability following electroporation b. Passing a small amount of culture into selective (and non-selective) broth allows for a relatively rapid assessment of the success or failure of transformation. Passing 50 μL into 1mL works well, though comparison to a blank is essential as the presence of dead cells can shift to pH downward in such a small volume.