Avoiding RNase contamination
Some guidelines on how to best avoid RNase contamination.
Common sources of contamination
- Buffers contaminated with microorganisms. (Note that the solutions need not be turbid to be problematic.)
- Pipettors. Keep a separate set of pipettors for RNA use to avoid contamination with RNases. Avoid touching the barrel or metal ejector to the sides of tubes.
- Keep separate a set of pipettors, glassware, plasticware, reagents, electrophoresis apparatus etc. for RNA use only.
- Store solutions in small aliquots and discard each aliquot after use.
- Clean electrophoresis apparatus.
- Wash with detergent solution.
- Rinse in H2O.
- Dry with ethanol.
- Fill with 3% solution of H2O2. (Don't use DEPC solutions because it will break down the plastic.)
- Incubate 10 mins at room temperature.
- Rinse with DEPC treated H2O.
- Handle chemicals with disposable spatulas.
- Whenever possible treat solutions with 0.1% DEPC for 1 hour at 37°C and autoclave 15 mins at 15 psi.
- Remove RNases from glassware by
- Bake glassware at 300°C for 4 hours or 180°C or higher for several hours.
- Alternatively, soak glassware in freshly prepared 0.1% (v/v) DEPC in water or ethanol for 1 hour, drain, and autoclave (necessary to destroy any unreacted DEPC which can otherwise react with other proteins and RNA).
- Rinsing with RNaseZap followed by two rinses with RNase free H2O.
- Sterile, disposable plasticware can safely be considered RNase-free and should be used when possible.
- Metal spatulas can quickly be decontaminated by holding in a burner flame for several seconds.
- Treat plasticware with RNAseZap or DEPC.
- Use RNase-free disposable tips and tubes. Use sterile forceps to transfer items to racks.
- Use gloves from a fresh box at all times. Don't touch the gloves to any surface that might be contaminated with RNases (like yourself, door handles, refrigerators, freezers etc.).
- According to tests run by Ambion, autoclaving H2O is sufficient to remove most (but not all) RNase activity from water.