How to make DEPC-treated water and Tris Buffer
|Date Added: Mon Feb 02 2009|
|Date Modified: Mon Feb 02 2009|
|Abstract: Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a strong, but not absolute, inhibitor of RNases. It is commonly used at a concentration of 0.1% to inactivate RNases on glass or plasticware or to create RNase-free solutions and water. DEPC inactivates RNases by covalent modification.|
Add 0.1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution.
Let the solution incubate for 12 hours at 37°C.
Autoclave for 15 minutes to remove any trace of DEPC.
How to make DEPC-treated Tris buffers: DEPC will react with primary amines and cannot be used directly to treat Tris buffers. DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO2. When preparing Tris buffers, treat water with DEPC first, and then dissolve Tris to make the appropriate buffer. Trace amounts of DEPC will modify purine residues in RNA by carboxymethylation. Carboxymethylated RNA is translated with very low efficiency in cell-free systems. However, its ability to form DNA:RNA or RNA:RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified. Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100°C for 15 minutes.