Which Water to Use? Avoid RNase contamination in reagents
by Sapna Chacko
- High quality, DEPC treated, and non-DEPC treated water
- Certified free of endonuclease, exonuclease, and RNase activity
- Available in sizes to suit all your experimental needs
RNase contamination in reagents used for RNA isolation and analysis can contribute to experimental inconsistency and sometimes even experimental failure. In trying to pinpoint the source(s) of RNase contamination, it is easy to overlook the water used in the experiment, either to prepare reagents, or to resuspend precipitated RNA. Even purified water can have a high pH and minerals that can interfere with reactions that require specific salt and pH conditions. Ambion offers three different grades of high quality water: DEPC-treated water, Nuclease-free water (not DEPC-treated) and RT-PCR grade water.
Ambion Certified and Guaranteed Nuclease-free
- DEPC-treated Water - DEPC destroys enzymatic activity by modifying -NH2, -SH and -OH groups in RNases and other proteins. DEPC treatment is a very effective way to treat solutions that will contact RNA. Ambion's DEPC-treated water is autoclaved both before and after packaging to ensure sterility and complete inactivation of DEPC.
- Nuclease-free Water (not DEPC-treated) - Ambion also offers nuclease-free water that is not DEPC-treated. Ambion's Nuclease-free Water is ideal for applications that are reported to be acutely sensitive to residual DEPC e.g., oocyte injection and other in vivo translation applications.
- RT-PCR Grade Water - Ambion's RT-PCR grade water is tested for prokaryotic as well as eukaryotic genomic DNA contamination using a sensitive end-point PCR assay based on detection of rRNA genes. Prokaryotic DNA contamination is assessed by performing PCR with 16S rRNA primers, and eukaryotic DNA contamination is assessed by performing PCR with 18S rRNA primers. Each lot is also tested for nuclease contamination. This product is ideal for use in any PCR or RT-PCR application.
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Inactivate RNases in Solution
The RNAsecure™ Reagent eliminates these problems. RNAsecure is a unique nonenzymatic reagent that will irreversibly inactivate RNases in solution. Unlike DEPC, RNAsecure can be used with virtually any solution and does not require post-treatment autoclaving. The inactivation process can be repeated at any time to protect against introduced contaminants. RNAsecure does not interfere with downstream procedures, such as RT-PCR and in vitro transcription, and is also compatible with Tris and other solutions that cannot be treated with DEPC.
The RNAsecure™ Resuspension Solution contains the same active ingredients as the RNAsecure Reagent, but is supplied at working concentration for direct resuspension of RNA pellets.
"By late 2004, I was experiencing problems with making transgenic fly stocks. I would prepare my transgene constructs the way I always had, make up injection mixes with them using standard protocols, and inject the constructs into appropriately staged D.melanogaster embryos. I would end up with <1% hatch rates and couldn't easily figure out why.
Knowing Ambion's Nuclease-free Water (non-DEPC-treated) to be some of the purest commercially available water, I purchased some for making my injection mixes. Over the time span of two weeks, I injected five transgenes, two of which had been injected previously at least twice without success. With Ambion water in my injection mixes, hatch rates were excellent, as were pre- and post-eclosion survival rates. Some six weeks after my injections, I have an average of 10 independent stocks for each of the five transgenes.
I'm happy to have found the solution to my transgene injection problem, and will be using Ambion water in my future injection experiments!"
DNaseAlert: Testing for DNases The DNaseAlert QC System is the ideal solution for the sensitive detection of a range of DNases (DNase I, micrococcal nuclease, mung bean nuclease, S1 nuclease, Bal31 nuclease, Benzonase, T7 endonuclease, and exonuclease III) in a simple, easy-to-use assay. Like RNaseAlert, DNaseAlert contains a unique DNA substrate tagged with both a fluorescent reporter and a dark quencher. When DNases are present, the linkage between the fluor and quencher is severed, which leads to the emission of a bright orange signal upon excitation. (Note: RNase activity can be measured simultaneously, using the RNaseAlert and DNaseAlert Kits.)