We have been following the conventional phenol chloroform method for plasmid isolation. At the same time we are also keen about the RNA bands on AGE. For this, we have omitted RNAse treatment at 60 C for 30 min. However, for small time period we are not getting RNA band on AGE although plasmid bands are clear.
We have changed the entire reagents, albeit no positive result.
What could be the crucial point for not getting RNA bands?
Have you grown the cultures for an unusually long period? Due to a hurricane warning I once had to leave small-scale cultures for 48 h in the shaker. When I returned I miniprepped them and, to my surprise, the plasmid was OK but tRNA was nowhere to be seen.
Another, more mundane possibility is someone adding RNAse to the cell resuspension solution and forgetting to warn everyone in the lab.
Hope it helps!
Many thanks Martin. Your answer is quite helpful. It is obvious that aged culture will less likely to havr RNA, may be its instability is one of the main points.
We have also used fresh culture but same result.
Hi Biswaranjan Paital
If your plasmid culture is ok, how much ng did you take for RNA synthesis?? Did you checked the concentration of RNA in the nanodrop?? another thing is that how much ng did you insert in the gel??
Thanks
We didn't synthesize RNA. Simple plasmid isolation in which we didnt notice RNA band but plasmid band was clear
Often the loading dye that people use for their gels has RNase added to it, in order to reduce the rRNA bands in plasmid mini preps. You might make sure that isn't the case with yours.
Thanks Michael, but the dye was prepared by us. We don't use RNAase into it.
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