Avoiding RNase contamination

Some guidelines on how to best avoid RNase contamination.

Common sources of contamination

1. Buffers contaminated with microorganisms. (Note that the solutions need not be turbid to be problematic.)
2. 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.

Precautions

1. Keep separate a set of pipettors, glassware, plasticware, reagents, electrophoresis apparatus etc. for RNA use only.
2. Store solutions in small aliquots and discard each aliquot after use.
3. Clean electrophoresis apparatus.
   1. Wash with detergent solution.
   2. Rinse in H₂O.
   3. Dry with ethanol.
   4. Fill with 3% solution of H₂O₂.
   5. Incubate 10 mins at room temperature.
   6. Rinse with DEPC treated H₂O.
4. Handle chemicals handled with disposable spatulas.
5. Whenever possible treat solutions with 0.1% DEPC for 1 hour at 37°C and autoclave 15 mins at 15 psi.
6. Bake glassware at 300°C for 4 hours.
7. Treat plasticware with RNAseZap or DEPC.
8. Use RNase-free disposable tips and tubes. Use sterile forceps to transfer items to racks.

Notes

• According to tests run by Ambion, autoclaving H₂O is sufficient to remove most (but not all) RNase activity from water. [1]

Reference

1. RNase and DEPC Treatment (This is a very good source with systematic studies of the effect of different variables on RNase activity.

2. Molecular Cloning

3. Ten Sources of RNase Contamination