Water for Nuclease-Free Applications

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.

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.

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

The ultrafilter comes with a certificate guaranteeing RNase-free water for three months when a BioPak point-of-use filter is connected to a high purity water purification system, and utilized in normal laboratory conditions.