Talk:IRPCM:Molecular Genetics:Transformation:Transformation Notes

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Transformation Notes: The transformation of the Mollicutes is anything but routine. There a couple of issues that need to be addressed before a successful transformation can be achieved. First, one needs to be sure that the concentration of the selective media is appropriate. By that, I mean that the level of antibiotics should be sufficient to eliminate background colonies and only that high. For instance, if 2 µg/ml tetracycline eliminates all background colonies, then don't use 10 µg/ml in your selective media. This has to be determined empirically for each species and each strain. If antibiotic concentrations are too high, you might not get any transformants to grow and would assume the worse. Second, not all antibiotics will be suitable for your species. Tetracycline seems to be the best first choice for testing, but gentamicin and other antibiotic markers have been used in mycoplasmas. Tetracycline seems to give the lost background in many species while gentamicin concentrations tend to be higher to achieve this result. Third, it goes without saying, that the selective media needs to be freshly made and that the organisms need to be cultivated under the optimal conditions for that species. Finally, there are two basic methods for transforming the Mollicutes, electroporation and PEG. Both methods require some trial and error to determine the optimal conditions. Don't assume that what works for one species will automatically work for all species. Plan to experiment with voltage, incubation conditions, PEG concentrations, etc. Working out the optimal conditions for your species will pay dividends down the road by saving time and money. I would try electroporation first. It seems to be the method of choice these days, but don't forget about the original PEG method. It might be useful with some species and transformation conditions.

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