Avoiding RNase contamination: Difference between revisions
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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.]] | |||
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. | ||
##Fill with 3% solution of H<sub>2</sub>O<sub>2</sub>. | ##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. | ||
#Handle chemicals | #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. | #Whenever possible treat solutions with 0.1% DEPC for 1 hour at 37°C and autoclave 15 mins at 15 psi. | ||
#Bake glassware at 300°C for 4 hours. | #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 H<sub>2</sub>O. | |||
#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. | #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. | ||
#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 H<sub>2</sub>O is sufficient to remove most (but not all) RNase activity from water. <cite>AmbionDEPC</cite> | |||
==Reference== | ==Reference== | ||
#[[Molecular Cloning]] | <biblio> | ||
#MolecularCloning [[Molecular Cloning]] | |||
#AmbionDEPC [http://www.ambion.com/techlib/tb/tb_178.html RNase and DEPC Treatment] (This is a very good source with systematic studies of the effect of different variables on RNase activity. | |||
#AmbionRNaseContamination [http://www.ambion.com/techlib/tn/93/9316.html Ten Sources of RNase Contamination] | |||
</biblio> | |||
==See also== | |||
* [[DEPC]] | |||
[[Category:RNA]] | [[Category:RNA]] |
Latest revision as of 09:39, 30 January 2009
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.
Precautions
- 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.).
Notes
- According to tests run by Ambion, autoclaving H2O is sufficient to remove most (but not all) RNase activity from water. [1]
Reference
-
RNase and DEPC Treatment (This is a very good source with systematic studies of the effect of different variables on RNase activity.