DEPC: Difference between revisions
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<small>(More DEPC is needed to inactivate larger amounts of RNases, but larger amount of RNases take longer to remove subsequently and increase the risk of residual DEPC, health risk, and interference with downstream applications. 0.05% is recommended, for example, in ''RNA methodologies'' by Farrell, whereas 1% is probably the most repeated number.)</small> | <small>(More DEPC is needed to inactivate larger amounts of RNases, but larger amount of RNases take longer to remove subsequently and increase the risk of residual DEPC, health risk, and interference with downstream applications. 0.05% is recommended, for example, in ''RNA methodologies'' by Farrell, whereas 1% is probably the most repeated number.)</small> | ||
*shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer | *shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer | ||
*DEPC must then be completely destroyed by autoclaving ( | *DEPC must then be completely destroyed by autoclaving (15-45min at 15psi on liquid cycle) | ||
<small>Protocols state very different autoclave times. If the fruity smell is faint, autoclaving was sufficient. Even after a long 45min autoclave treatment the smell will not be completely gone. If you use more than 0.05% and have a very sensitive downstream experiment, it does not hurt to autoclave longer or twice.</small> | |||
==Warnings== | ==Warnings== | ||
*DEPC in unstable in aqueous solutions. It hydrolyses quickly to CO<sub>2</sub> and ethanol. Half-life in phosphate buffer at pH 6 is 20min. | *DEPC in unstable in aqueous solutions. It hydrolyses quickly to CO<sub>2</sub> and ethanol. Half-life in phosphate buffer at pH 6 is 20min. | ||
[[Image:Fruits.jpg|thumb|DEPC's fruity smell|right]] | |||
*Nucleophiles greatly speed up DEPC hydrolysis and are consumed in the process. Therefore, DEPC cannot be used with solutions that contain nucleophiles such as Tris (amines) and HEPES (amines) buffers or mercaptans (thiol). In such cases, use DEPC-treated water to generate the solution. | *Nucleophiles greatly speed up DEPC hydrolysis and are consumed in the process. Therefore, DEPC cannot be used with solutions that contain nucleophiles such as Tris (amines) and HEPES (amines) buffers or mercaptans (thiol). In such cases, use DEPC-treated water to generate the solution. | ||
*Although H<sub>2</sub>O prepared with reverse osmosis systems like Milli-Q is free of RNases [http://www.ncbi.nlm.nih.gov/pubmed/8643347 (Huang 1995)], it can be contaminated by microbial growth if the machine and piping are not well maintained. This is especially problematic in centralised water systems with metres of piping. | *Although H<sub>2</sub>O prepared with reverse osmosis systems like Milli-Q is free of RNases [http://www.ncbi.nlm.nih.gov/pubmed/8643347 (Huang 1995)], it can be contaminated by microbial growth if the machine and piping are not well maintained. This is especially problematic in centralised water systems with metres of piping. | ||
*The fruity smell coming from ageing DEPC-containing solutions stems from the esters that are generated when ethanol produced from hydrolysis reacts with residual carboxylic esters. It is normal that some fruity smell lingers after DEPC removal and is not necessarily a sign of incomplete DEPC hydrolysis. | |||
==Effect of residual DEPC on RNA== | ==Effect of residual DEPC on RNA== | ||
Latest revision as of 06:59, 15 April 2009
Diethyl pyrocarbonate (DEPC) is an efficient, nonspecific inhibitor of RNases. It is typically used to treat water and solutions before working with easily degraded RNA. DEPC reacts with amine, hydroxy and thiol groups of proteins thereby inactivating RNAses (and other enzymes).
Procurement
- 100ml DEPC from Sigma (D5758) [1] - €233/$292 (as of 2009-01)
- 100ml DEPC from MPI (150902) [2] - €313 (as of 2009-01)
DEPC-treatment of solutions
- add 0.05-0.1% (v/v) DEPC to solution/water
(More DEPC is needed to inactivate larger amounts of RNases, but larger amount of RNases take longer to remove subsequently and increase the risk of residual DEPC, health risk, and interference with downstream applications. 0.05% is recommended, for example, in RNA methodologies by Farrell, whereas 1% is probably the most repeated number.)
- shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer
- DEPC must then be completely destroyed by autoclaving (15-45min at 15psi on liquid cycle)
Protocols state very different autoclave times. If the fruity smell is faint, autoclaving was sufficient. Even after a long 45min autoclave treatment the smell will not be completely gone. If you use more than 0.05% and have a very sensitive downstream experiment, it does not hurt to autoclave longer or twice.
Warnings
- DEPC in unstable in aqueous solutions. It hydrolyses quickly to CO2 and ethanol. Half-life in phosphate buffer at pH 6 is 20min.
- Nucleophiles greatly speed up DEPC hydrolysis and are consumed in the process. Therefore, DEPC cannot be used with solutions that contain nucleophiles such as Tris (amines) and HEPES (amines) buffers or mercaptans (thiol). In such cases, use DEPC-treated water to generate the solution.
- Although H2O prepared with reverse osmosis systems like Milli-Q is free of RNases (Huang 1995), it can be contaminated by microbial growth if the machine and piping are not well maintained. This is especially problematic in centralised water systems with metres of piping.
- The fruity smell coming from ageing DEPC-containing solutions stems from the esters that are generated when ethanol produced from hydrolysis reacts with residual carboxylic esters. It is normal that some fruity smell lingers after DEPC removal and is not necessarily a sign of incomplete DEPC hydrolysis.
Effect of residual DEPC on RNA
Traces of DEPC modify purine residues (A+G) in RNA by carboxymethylation. Therefore, DEPC must always be removed from solutions or containers by autoclaving or heating at 100°C for 15 min. Cell-free translation of carboxymethylated RNA will yield less protein than with unmodified template. Hybridisation is not seriously affected unless the RNA probe is heavily carboxymethylated.
Safety
- DEPC is carcinogenic (it carboxymethylates purines) and should be handled with care. Wear gloves!
- Di-methyl-propyl carbonate (DMPC), a safer alternative to DEPC, is used in exactly the same way.
Internal links
External links
- Handling RNA from Stanley lab, Yeshiva Uni, NY
- RNase and DEPC Treatment: Fact or Laboratory Myth
- Molecular biology grade DEPC from Sigma
- Molecule of the day: DEPC - Making the world safe for RNA everywhere) ;-)
- DEPC at PubChem
- DEPC at wikipedia
DEPC-related discussion threads:
