Dear
I'm using CTAB /chloroform method for extracting RNA from a plant material with potential problem of polysaccharide. Sometimes I successfully extracted the RNA (with obvious DNA) with clear 28s and 18s bands (sometimes with 5s). However, most times I got only sharp DNA band without 28s and 18s bands. Although I guess that RNA might be degraded but I do not observed smear in the gel (And I always got very clear DNA band). Would anyone know how could I improve my RNA extraction procedure?
Hello
I guess if RNA is already degraded, it is most likely those are removed the ethanol/isopropanol precipitation or washing step (or most of them may be too small to see on the gel).
For example, DNA precipitate when >2.0 volume of ethanol added to the solution, whereas RNA precipitation requires >2.5 volume of ethanol. Adding some salt may improve the precipitation too. At the same time, maybe better use > 80% ethanol at the pellet washing step rather than 70% ethanol. If it still does not work, I may use column instead of precipitation methods, or change the stating material. We use Trizol for RNA prep and most of the time it work well for us. We extended the lysate step for ~1 hour instead of 10 minutes described in the original protocol.
Good luck with your extractions.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts