I am working on an EMCV like virus with about 8kbp genome. The RNA is usually prepared from digested plasmids by in-vitro transcription, followed by DNase I digestion, followed by P/C and Ethanol cleanup and finally electroporated into Hela cells. However, due to the large fragile nature of this genome, it is hard to get the complete sequence in the cells without it falling appart, and often it has a low read, even if it showed good bands on a gel run on the same day. So I was thinking and wanted to try skipping the purification procedures all together and just adding EDTA to bind the magnesium and then electroporating. Do you think it would be a good idea? Any other tips and tricks for this problem? Thanks!
lots of/more salts in the in vitro transcription buffer, the electric current will be higher, the cells might die and a Short circuit may occur.
I am doing a PRRSV reverse genetic system, the purification seems ok.
Hi
It is correct. The viral RNA after undergoing molecular procedures will have a lot of components. Particularly salts will create problems with low time constant during electroporation, and the instrument may also get damaged. Better to purify it and handle with gentle procedures recommended in literature for handling large nucleic acid molecules
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