Water for Nuclease-Free Applications
Impact of Water
Ultrafiltration is a membrane-based technology that removes nucleases by a filtration process. Ultrafilters are depth filters with nominal weight cut-off ranging from 5 000 Da to 100 000 Da. To remove nucleases, filters with a molecular weight from 5 KDa to 13 KDa are selected.
Ultrafilters are positioned at the point-of-use, at the outlet of the water purification process. The water delivered is RNase-free and does not require DEPC treatment.
Data shown on Figure 1 demonstrate the efficiency of RNase removal by a 13 KDa ultrafilter (BioPak®). Ultrafiltration is fully efficient over a three month period and large quantities of nucleases.
Figure 1. Pure water is spiked with RNase (RNase A, Sigma) and the solution is filtered through an ultrafiltration cartridge (BioPak®). The RNase concentration is measured (kit RNase alert kit, Ambion AM1966) at the inlet and at the outlet of the ultrafiltration cartridge. (Click on image to enlarge)
Figure 2 highlights the lifetime of rRNA prepared in DEPC treated water and water purified using ultrafiltration. The rRNA stability in water purified using ultrafiltration compares well with stability in DEPC-treated water.
Figure 2. Stability (4 °C) of rRNA prepared in water purified by ultrafiltration (Milli-Q® UF) and DEPC treated water. (Click on image to enlarge)
Conclusions
- Ultrafiltration is an alternative to DEPC treatment to obtain RNase-free water.
- It presents some advantages over the typical DEPC procedure:
- Easier: water delivered by a high purity water purification system equipped with an ultrafiltration cartridge is RNase-free
- Safer: DEPC is a suspected carcinogen
- Faster: no need to proceed to the DEPC treatment (typically one hour)
- Maintains the water quality
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