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BARNSTEAD’S TECHNICAL APPLICATION BULLETIN T

BULLETIN 0012

. V

ALIDATIONOF Nucleases (Ribonuclease and Deoxyribonuclease)

A

. NANO

PURE ®

DI

AMOND

B

.

™

UV/UF W

ATER

A NANOpure DIamond UV/UF (D11931) was set upand installed with an Organic Free Cartridge Pack (D50281). An “air purge” of the cartridge pack was performed with the menu selection, and then 10 L of water was dispensed to drain to rinse and wet the 0.2 µm hollow-fiber final filter. Next, a system sanitization was performed. The sanitization cycle is software driven – the analyst simply needs to inject a liquid sanitant provided in a syringe (CMX25) and allow the cycle to run for 4 hours. The cycle sanitizes the ultrafilter and the system tubing up to the point of use. After the sanitiza- ton cycle the system was allowed to operate for two days in order to fully equilibrate the resins and ultrafilter. In this nor- mal mode of operation the system continuously recirculates pure water.

T

he nucleases - ribonculease (RNase) and deoxyri-bonuclease (DNase) - are ubiquitous contaminantsin the laboratory environment. Glassware and solu-tions must be rendered nuclease-free when working withRNA or DNA to ensure good recoveries. Therefore, it iscritical that ultrapure water used for reagent preparationand glassware rinsing is free of nucleases. In this valida-tion study, a NANOpure DIamond UV/UF ultrapure watersystem was spiked with feedwater containing high levels ofRNase and DNase (in two separate experiments) and theproduct water was analyzed for their presence. The exper-iments were performed by Mo Bio Laboratories, Inc. inSolana Beach, CA. The results indicate that the DIamond™UV/UF effectively removed the nucleases from the water tolevels below the sensitivity limit of the test. abcdefghi

I NTRODUCTION

Nucleases are small enzymes present in all cell types and are responsible for the destruction of RNA and DNA. They areresistant to heating, are active over a wide pH range, renaturereadily, and can easily be transferred with an analyst’s fin-gers. Consequently, nucleases are found on most laboratoryglassware and in most solutions. Traditionally, a 0.1% solu-tion of diethyl pyrocarbonate (DEPC) has been used to inac-tivate nucleases in buffers and water. However, since DEPCis a nonspecific inhibitor, it must be removed from treatedsolutions before being used with nucleic acids. This is typi- cally done by autoclaving at 70°C for 1 hour. DEPC treat- ment is not only time consuming, it is also toxic. A point of use ultrapure water system that can produce nuclease free water without DEPC treatment can be a valuable piece ofequipment in the laboratory for saving time and producingquality results. F

IGURE

1.

Gel electrophoresis results for RNase. The identity of the lanes is as follows: a) blank, b) 2.0 L sample, c) 2.5 L sample, d) 5.0 L sample, e) 10.0 L sample, f) 20.0L pooled sample, g) negative control, h) low-level positivecontrol, i) high-level positive control.

RN ASE A NALYSIS

Spike solutions. RNase is measured in Kunitz units (Ku)which is the amount of RNase that completely hydrolizes 1 µg of RNA in 15 minutes at 37°C. A 500 ml spike solution with a concentration of 9.00x10

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Ku/µL was prepared from USBiologicals RNase A. If this 500 mL spike were diluted to 20 L a 2.25x10

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Ku/µL solution would result which is well abovethe 10

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Ku/µL sensitivity of the analysis. The intent was tocollect a 20 L pooled sample from the system after spiking, which would be above the sensitivity of the analysis if the spike broke through the system.

HE U LTRAPURE W ATER S YSTEM

Positive controls. An undiluted spike was used as a high-level positive control (9.00x10 The NANOpure DIamond UV/UF water system uses carbonadsorption, ion exchange, UV oxidation, and ultrafiltration (UF) to produce ultrapure water. This serial approach ensures the removal of a broad range of contaminants found in water. It is likely that each of these technologies is involved with the removal of nucleases. In this study, a NANOpure DIamond UV/UF was spiked with RNase and DNase in two separate experiments and the product water was evaluated for RNase and DNase activity using gel elec- trophoresis. The analysis was performed by Mo Bio Laboratories in Solana Beach, CA.

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Ku/µL) and a 20x dilutionwas used as a low-level positive control (2.25x10

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Ku/µL). Negative control. A negative control was prepared by treat-ing deionized water with DEPC.