I know the methods available for transforming bacteria in order to take up foreign plasmid DNA. I, however, would like to utilize a non-viral method to infect my bacteria with foreign RNAs. I have been unable to locate a protocol that explicitly mentions its application for this matter and am uncertain if using chemically competent E. coli or electroporation will succeed in depositing foreign ssRNAs into my cells. If anyone has any knowledge on how to do this any and all help would be appreciated.
I should note that I have a means of doing this by using a thermally sensitive plasmid (due to the pSC101 derived ORI101) that should degrade at 42 degrees and while the sense RNA remains intact. This method has, however, behaved in an undependable manner thus far, so I could use another method in the mean time in order to collect my data.
Thank you!
Hi ! Yes there are. Electroporation is one of them.
Cacl2 treated cell and Lysozyme- EDTA spheroplast can also be used but it is less efficient and intracellular RNAse activity may be an issue with the last two methods I mentioned. Go with electroporation with single shock at 6.25 kV/em with a 25 pF capacitor.
If RNA infectivity yield is low, increase it by adding sodium salts of citrate, phosphate and sulfate (each 1 raM) to the eletroporation medium. I hope it helps.
Ritu Rathi
I had read a paper by Akira Taketo that had similar advisements, but found his brief language somewhat confusing. At what point should I add the sodium salts? I have read elsewhere that the electroporation cell suspension must have low salt concentrations to prevent arcs from the electricity.
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