|
| |
|
|
| DIETHYL PYROCARBONATE (DEPC) TREATMENT OF AQUEOUS SOLUTIONS
|
| [ 文章来源：omrf.ouhsc.edu/~frank | 文章作者：
| 发布时间：2007-01-09|
字体：
[大
中
小]
| |
|
Diethyl pyrocarbonate derivitizes histidine residues and is therefore an effective method to inactivate nucleases including RNAse. CAUTION: DEPC is irritating to the eyes, skin, and mucous membranes. It is a suspected carcinogen. It hydrolitically decomposes to CO2 & ethanol and may decompose to urethane (a possible carcinogen) in the presence of ammonia. Use it in the fume hood and wear gloves! DEPC is highly unstable in Tris buffers so avoid use of TE for DNA dilutions prior to DEPC treatment whenever possible. The CO2 generated during the decomposition of DEPC may reduce the pH of the solution. It is therefore best to EtOH precipitate the DNA and redissolve it in the appropriate buffer before further manipulations are undertaken.
- Use DEPC only from a recently opened container (one month old or less). Be sure to wear gloves when working with the DEPC, and open it only in the fume hood. Due to build up of pressure or faulty glass, Sigma has reported bottles of DEPC have exploded!
- Dilute the DEPC 1:100 in absolute ethanol.
- For 1X DEPC treatment, add 1/10 volume of the 1:100 diluted DEPC (from step 2) to 1 volume of solution to be treated. Thus, the working concentration of DEPC is 0.1% v/v. For DNA treatment, the total volume should be at least 100µl to avoid evaporation in the next step. Plasmid preps may need a 3-4x DEPC treatment (0.3 - 0.4% by volume).
- Vortex and incubate at 65°C for one hour WITH THE CAP OFF THE MICROFUGE TUBE to release the CO
2 that will be generated. The reaction is complete when bubbles stop forming in the tube. For treatment of large aqueous volumes, incubate at 37-42°C overnight followed by 45 minutes of autoclaving. The DEPC smell should be gone.
上一篇：How to make DEPC-treated water and Tris Buffer 下一篇：RNase Protection Assay--RNA酶保护
|
|
|
How to make DEPC-treated