DEPC: Difference between revisions
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*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. | |||
==Effect of residual DEPC on RNA== | ==Effect of residual DEPC on RNA== | ||
Revision as of 11:11, 29 January 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
(Many protocols recommend 0.1% but 0.05% works just as well and is easier to remove subsequently.)
- shake for 1h to O/N on orbital shaker OR 20-30min with a magnetic stirrer
- DEPC must then be completely destroyed by autoclaving (15min at 15psi on liquid cycle)
(Esters may be generated during autoclaving giving rise to a 'fruity' smell (that is not coming directly from DEPC).
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.
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
- Comparison of DEPC-treated water and Milli-Q PF Plus water (similar RNA degradation in Milli-Q and DEPC-treated water)
- Molecule of the day: DEPC - Making the world safe for RNA everywhere) ;-)
DEPC-related discussion threads:
