DEPC Water
From Protists
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==DEPC Water Protocol== | ==DEPC Water Protocol== | ||
*Check and see if we have DEPC in the 4C drawer (be careful of built up pressure/explosion and only work with DEPC in the hood with appropriate safety protection); ''Note:'' DEPC is available at CSS with the catalog number 275755 for ~$36/5mL | *Check and see if we have DEPC in the 4C drawer (be careful of built up pressure/explosion and only work with DEPC in the hood with appropriate safety protection); ''Note:'' DEPC is available at CSS with the catalog number 275755 for ~$36/5mL | ||
| - | *make a 0.1% v/v ratio of DEPC to distilled water (ex. | + | *make a 0.1% v/v ratio of DEPC to distilled water (ex. 40uL DEPC into 40mL dH2O; 500uL into 500mL dH2O) |
*[[Autoclave]] on liquid cycle for appropriate amount of time based on volume of DEPC Water made | *[[Autoclave]] on liquid cycle for appropriate amount of time based on volume of DEPC Water made | ||
*Allow to cool and store in 4C or aliquot into RNase-free eppendorf tubes in the -20C for future use | *Allow to cool and store in 4C or aliquot into RNase-free eppendorf tubes in the -20C for future use | ||
Current revision as of 15:53, 9 March 2009
Contents |
Background
- DEPC = diethylpyrocarbonate
- adding DEPC to water is a standard preventative measure when RNase-free conditions are required (when dealing with RNA)
- DEPC is an alkylating reagent that binds to histidine residues of the catalytic site of RNases, which destroys their enzymatic activities
- However, DEPC is bad because:
- incomplete removal of DEPC results in carboxymethylation of purine residues in RNA or single stranded DNA (ssDNA), causing a loss of biological activity
- it reacts with compounds with primary amine groups, such as Tris (EB buffer from Qiagen kits is Tris buffer)
- it introduces high levels of organics and ions to the solution
- Ribonucleases (RNAses) are enzymes that catalyze the degredation of RNA molecules (they can withstand temperatures up to 100C, extreme pH and remain active for weeks; they are also ubiquitous since microorganisms in air, on surfaces or in the water supply are typical environmental sources, but they are also present on human skin and in body fluids)
- DEPC Water is NOT recommended for use in PCR, sequencing or in vivo experiments
- DEPC should be stored at 2-8C because it can become pressurized by reacting with water molecules in the air and can cause explosions
DEPC Water Protocol
- Check and see if we have DEPC in the 4C drawer (be careful of built up pressure/explosion and only work with DEPC in the hood with appropriate safety protection); Note: DEPC is available at CSS with the catalog number 275755 for ~$36/5mL
- make a 0.1% v/v ratio of DEPC to distilled water (ex. 40uL DEPC into 40mL dH2O; 500uL into 500mL dH2O)
- Autoclave on liquid cycle for appropriate amount of time based on volume of DEPC Water made
- Allow to cool and store in 4C or aliquot into RNase-free eppendorf tubes in the -20C for future use
How to Dispose of DEPC After Use
- autoclave the DEPC solution at 121C for at least 60min
- this causes the DEPC to be converted into ethanol and CO2, which are left as contaminants in the water
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