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Water for Nuclease-Free Applications

 
 
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Application Overview


Nuclease-free water is used in assays and experiments involving nucleic acids. Nucleases include DNases, specific for DNA degradation, and RNases, specific for RNA degradation. These enzymes can degrade the nucleic acids from an extremity of the strand or from the middle of the strand, and are called respectively exonucleases and endonucleases. In situ, RNases degrade foreign nucleic acids, regulate gene expression by degrading mRNA, have some antitumor activity and mature various RNA.

Several classes of these enzymes exist in bacteria, with various selectivity and activity. In water, nucleases originate from bacteria, and the molecular weight of the bacterial RNases may vary significantly. For instance RNAse I has a molecular weight of 27 000 Da, while RNase III has two 26 500 Da subunits.

These enzymes are extremely active and a small amount of those nucleases will generate the degradation of the nucleic acids present in the samples. It is therefore particularly important to keep the sample, buffers, plasticware and glassware, benches, and all containers free of nucleases at all times. Water, as the major component of the buffers utilized for the assays should be nuclease-free.

Nuclease-free water has typically been prepared via DEPC (diethylpyrocarbonate) treatment of the buffers. Enzymes are inactivated during the process, as the nitrogen atoms from histidine residues of the RNase active site reacts with the DEPC. Because the nitrogen reacts with DEPC, buffers based on amine containing compounds, such as HEPES and Tris cannot undergo DEPC treatment. Following the inactivation step, the excess of DEPC is eliminated by a 1 h autoclaving step.

By-products of the chemical inactivation, as well as by-products generated during the autoclaving include ethanol and CO2, which reacts with water to generate carbonic acid and bicarbonate. Therefore, both DEPC-treatment by-products impair the water quality: ethanol contributes to the TOC, and bicarbonate increases the conductivity.

Merck:/Freestyle/LW-Lab-Water/applications/Nuclease-Free Applications/LW-LC-NFApp-Image1-460x100.jpg
Figure 1: Degradation of DEPC yields to water contamination



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