Which Water to Use?

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

All Ambion water products are Type II 18 ohm filtered, and are certified to be nuclease-free. Our water undergoes state-of-the-art reverse osmosis filtration and deionization, followed by autoclaving and sterile filtration. Ambion's water purification equipment is subjected to routine maintenance and testing procedures according to rigid ISO 9001 specifications. Finally, in order to be certified nuclease-free, each lot of water is tested for nuclease activity by incubating a sample overnight with a radiolabeled RNA or DNA probe. After incubation, the probe is run on a gel to detect any degradation from nuclease.

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.

Bulk and Custom Orders

All of Ambion's water products can be packaged into larger custom sizes to fit your needs. Contact us at custom@ambion.com for more information.

Inactivate RNases in Solution

DEPC treatment of solutions that come in contact with RNA can be time-consuming, possibly hazardous, and only eliminates RNases present at the time of treatment. Some solutions (e.g., primary amine containing compounds such as Tris) cannot be treated with DEPC at all.

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.

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Water Determined Success of Drosophila Transgene Experiments Martin Kracklauer, PhD student in Dr. Janice Fischer’s laboratory at the University of Texas writes:

"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!"

Test for RNase and DNase Contamination This patent-pending technology detects RNase activity in a convenient and sensitive assay that delivers fast, accurate results in real time. The RNaseAlert Kit uses a novel RNA substrate tagged with a fluorescent reporter molecule (fluor) on one end and a quencher on the other. When RNases are present, the RNA substrate is cleaved and the fluor and quencher adducts are spatially separated in solution. This causes the fluor to emit a bright green signal that can be readily detected upon excitation at the appropriate wavelength. The sequence of the RNaseAlert Substrate is optimized to detect RNase A, RNase T1, RNase I, micrococcal nuclease, S1 nuclease, mung bean nuclease, and Benzonase®. (Note: DNase activity can be measured simultaneously, using the RNaseAlert and DNaseAlert Kits.)

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.)