Last time when I was extracting RNA using the TRIzol method, I accidentally washed the RNA precipitation with 25% ethanol (rather than 75%), then the precipitation fully dissolved. As they were important samples, I tried to re-pricipitate them by adding 5M RNase-free ammonium acetate, and added absolute ethanol to ~80% concentration. After re-precipitation ,washing by 80% ethanol and reconstitution, I got RNA solution with normal concentration, standard A260/280 ratio (2.0-2.2) but high A260/230 ratio (~2.3). I repeated this procedure several times and such things always happen. So I wonder why I got this kind of A260/230 ratio, is there any contamination or it merely meant that my RNA sample was ultra pure? (or maybe the RNA chain broke due to too much washing steps?) Thanks a lot!
Yifei Ding Our lab consider RNA A260/230 from 2.0 to ~2.5 is acceptable, and you showed that A260/280 was in normal range, therefore I don't think there's contamination. Also we sometimes repeat EtOH washing step just to obtain a better purity, just by adding reagents or centrifugation shouldn't cause significant damage to RNA sample.
as far as I know, A260/230 ratio is used to indicate the presence of unwanted organic compounds such as Trizol, phenol, Guanidine HCL, and guanidine thiocyanate.
so as you say it is degraded RNA it is not possible.
further, you can run an agarose gel to test the integrity of your purified RNA
all the best
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts