Estelle Riche, Stephane Mabic
Millipore S.A.S, Lab Water, Saint-Quentin-en-Yvelines, France
Nuclease-free glassware and reagents are required for many molecular biology applications. Chemical treatment with diethylpyrocarbonate (DEPC) has long been a common practice to generate nuclease-free water, but this method has several drawbacks. Alternatively, an ultrafiltration cartridge can be placed at the outlet of a water purification system in order to deliver nuclease-free ultrapure water that can directly be used for experiments.
Nucleases degrade DNA and RNA by cleaving the phosphodiester bonds of the nucleic acid backbone. While some nucleases are useful research tools, they are often a concern for scientists looking to preserve sample integrity for applications such as PCR, cloning, sequencing, or gene editing. For this reason, scientists working with nucleic acids require nuclease-free reagents and containers. Water, as the major component of buffers and reagents, should also be nuclease-free. Unfortunately, nucleases are ubiquitous in the laboratory: they are commonly present on plasticware and in reagents, and may even originate from the scientists themselves (skin, saliva, etc.).1 They are also very stable and difficult to inactivate;2 therefore, DEPC treatment is often used to eliminate them from solutions. Ultrafiltration is presented here as a convenient and safe alternative to DEPC treatment for the preparation of nuclease-free water.
Nuclease contamination of reagents used in molecular biology, and particularly water, may lead to inconsistent results or loss of valuable samples. DEPC is an efficient, nonspecific inhibitor of RNase and has been used for many years to inactivate RNases.3 Chemical treatment of solutions with DEPC is an efficient method, but it presents several drawbacks:
Figure 1.Degradation of DEPC releases contaminants into water.
Ultrafiltration was tested as an alternative method to generate nuclease-free water. This process uses hollow fibers to separate contaminants based on their size. A disposable cartridge containing 13 000 Da nominal molecular weight limit (NMWL) cut-off polysulfone ultrafiltration fibers was tested (Figure 2).
Figure 2.Polysulfone hollow fiber used for ultrafiltration in the Biopak® cartridge.
Figure 3 shows that the rRNA remained intact when the RNase solution was first filtered using ultrafiltration. In comparison, conventional DEPC treatment of the RNase solution similarly prevented rRNA degradation. As a control, when the RNase solution was left untreated, rRNA was digested. This demonstrates that the efficiency of RNase removal by ultrafiltration is equivalent to the inactivation of RNase activity by DEPC treatment.5
Figure 3.Gel electrophoresis of rRNA in water previously spiked with RNase and either DEPC-treated, ultrafiltered, or left untreated.
Ultrapure water was spiked with RNase A and divided into three aliquots. The solutions were either (1) treated with DEPC and autoclaved; (2) treated with ultrafiltration through a Biopak® ultrafiltration polisher; (3) not treated. Ribosomal RNA was added to each solution, incubated for 20 minutes, and then agarose gel electrophoresis in denaturing conditions was performed.
Nuclease-free water is required for many molecular biology applications. Unlike DEPC treatment, which is time-consuming and cumbersome, using an ultrafiltration cartridge is a safe and convenient way to obtain nuclease-free water from a water purification system.
This method has many benefits for scientists:
The authors are grateful to Julien Bole and Ichiro Kano for their technical expertise.
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