I have extracted the plasmid with the Qiagen maxi Endofree kit. the result of gel electrophoresis for this sample showed a band of degraded RNAs. Now, I want to remove RNAs from the eluted plasmid dissolved in WFI (I don't like extract it again). I think I have to precipitate the plasmid, RNase A treatment, and pass it through the resin column to remove impurities like RNase A and RNAs. I need the eluted plasmid for transfection and transduction of Jurkat cells. is there any protocol with details? how can I do it?
First of all the RNA is usually not a problem and shouldn't interfere with transfection or transduction. Unless you have a reason to clean it up, I would just ignore it.
The one thing it complicates is if you are quantifying your DNA by A260 then the RNA will also absorb and give you an incorrect reading. However treating with RNase will just convert the RNA to nucleotides and those will still absorb unless you purify.
thanks, Michael J. Benedik
ya, RNA makes a mistake in the reading DNA absorbance (A260) thus it affects the use of the correct concentration of DNA in transfection optimization (DNA: reagent ratio). so that I have to use a small amount of DNA and a lot of reagents that are toxic for the cells, I prefer to treat the eluted plasmid with RNase A and then purify it by the resin column.
unfortunately, I haven't found a protocol with details for this. I have just found a protocol for RNase A treatment (and not subsequent steps).
You could just do an ethanol precipitation after the RNase treatment. Or you can assess your DNA concentration by gel (against knowns standards) and just use that value.
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