Avoiding RNase contamination

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##Bake glassware at 300°C for 4 hours or 180°C or higher for several hours.
##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).
##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 H<sub>2</sub>O.
#Sterile, disposable plasticware can safely be considered RNase-free and should be used when possible.
#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.
#Metal spatulas can quickly be decontaminated by holding in a burner flame for several seconds.

Revision as of 16:25, 11 December 2006

Some guidelines on how to best avoid RNase contamination.

Contents

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. (Don't use DEPC solutions because it will break down the plastic.
    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. Remove RNases from glassware by
    1. Bake glassware at 300°C for 4 hours or 180°C or higher for several hours.
    2. 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).
    3. Rinsing with RNaseZap followed by two rinses with RNase free H2O.
  7. Sterile, disposable plasticware can safely be considered RNase-free and should be used when possible.
  8. Metal spatulas can quickly be decontaminated by holding in a burner flame for several seconds.
  9. Treat plasticware with RNAseZap or DEPC.
  10. 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. [AmbionDEPC]
  2. Molecular Cloning [MolecularCloning]
  3. Ten Sources of RNase Contamination [AmbionRNaseContamination]
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