DEPC: Difference between revisions
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*Note that DEPC cannot be used with chemical solutions that have amine groups, such as Tris and HEPES buffers, or mercaptans. In such cases, use DEPC-treated water to generate the solution. | *Note that DEPC cannot be used with chemical solutions that have amine groups, such as Tris and HEPES buffers, or mercaptans. In such cases, use DEPC-treated water to generate the solution. | ||
==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== | ==Safety== | ||
Revision as of 10:41, 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)
Use
- Treatment involves adding DEPC to 0.1% v/v and incubating at 37°C for 1 hour to overnight followed by autoclaving. Autoclaving destroys DEPC and is an essential step. Esters may be generated during autoclaving giving rise to a 'fruity' smell (that is not coming directly from DEPC).
- Note that DEPC cannot be used with chemical solutions that have amine groups, such as Tris and HEPES buffers, or mercaptans. In such cases, use DEPC-treated water to generate the solution.
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)
DEPC-related discussion threads:
