Avoiding RNase contamination

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[[Image:Scary-RNase-A.png|right|thumb|150px|Especially RNase A (structure on top) is scary for RNAs and RNA researchers.]]
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Some guidelines on how to best avoid RNase contamination.
Some guidelines on how to best avoid RNase contamination.
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##Rinse in H<sub>2</sub>O.
##Rinse in H<sub>2</sub>O.
##Dry with ethanol.
##Dry with ethanol.
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##Fill with 3% solution of H<sub>2</sub>O<sub>2</sub>.
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##Fill with 3% solution of H<sub>2</sub>O<sub>2</sub>. (Don't use DEPC solutions because it will break down the plastic.)
##Incubate 10 mins at room temperature.
##Incubate 10 mins at room temperature.
##Rinse with DEPC treated H<sub>2</sub>O.
##Rinse with DEPC treated H<sub>2</sub>O.
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#Handle chemicals handled with disposable spatulas.   
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#Handle chemicals with disposable spatulas.   
#Whenever possible treat solutions with 0.1% DEPC for 1 hour at 37&deg;C and autoclave 15 mins at 15 psi.
#Whenever possible treat solutions with 0.1% DEPC for 1 hour at 37&deg;C and autoclave 15 mins at 15 psi.
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#Bake glassware at 300&deg;C for 4 hours.
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#Remove RNases from glassware by
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##Bake glassware at 300&deg;C for 4 hours or 180&deg;C or higher for several hours.
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##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).
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##Rinsing with RNaseZap followed by two rinses with RNase free H<sub>2</sub>O.
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#Sterile, disposable plasticware can safely be considered RNase-free and should be used when possible.
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#Metal spatulas can quickly be decontaminated by holding in a burner flame for several seconds.
#Treat plasticware with RNAseZap or DEPC.
#Treat plasticware with RNAseZap or DEPC.
#Use RNase-free disposable tips and tubes.  Use sterile forceps to transfer items to racks.
#Use RNase-free disposable tips and tubes.  Use sterile forceps to transfer items to racks.
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#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.).
==Notes==
==Notes==
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#AmbionRNaseContamination [http://www.ambion.com/techlib/tn/93/9316.html Ten Sources of RNase Contamination]
#AmbionRNaseContamination [http://www.ambion.com/techlib/tn/93/9316.html Ten Sources of RNase Contamination]
</biblio>
</biblio>
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==See also==
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* [[DEPC]]
[[Category:RNA]]
[[Category:RNA]]

Current revision

Especially RNase A (structure on top) is scary for RNAs and RNA researchers.
Especially RNase A (structure on top) is scary for RNAs and RNA researchers.

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 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.
  11. 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.).

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]

See also

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