Qiagen, Y9370L, Thermostable Pyrophosphatase (1250 U)

1250 U of Thermostable Pyrophosphatase (0.625 mL at 2,000 U/mL)

Features

- Hydrolysis of inorganic pyrophosphate to produce orthophosphate
- Low K m (5.4 µM)
- Active at pH 7–9
- Optimal temperature for activity at 75°C

Product Details

Thermostable pyrophosphatase is a recombinant enzyme from Sulfolobus acidocaldarius , which catalyzes the Mg 2+ -dependent reaction of P 2 O 7 -4 + H 2 O → 2HPO 4 -2 . It has a low K m (5.4 µM) for pyrophosphate, is active between pH 7 and 9, has an optimal temperature for activity at 75°C and is functional under PCR conditions (1-3).

Supplied in: 10 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol (pH 7.5 at 25°C)

Performance

Test: Amount tested
Purity: n/a
Specific activity: n/a
Single-stranded exonuclease: 50 U
Double-stranded exonuclease: 50 U
Double-stranded endonuclease: 50 U
E. coli DNA contamination: 50 U

- Storage temperature: –25°C to –15°C
- Molecular weight: 19.4 kDa

Principle

The protein is produced by an E. coli strain carrying the Thermostable Pyrophosphatase gene from S. acidocaldarius .

One unit is the amount of enzyme that will liberate 1 µmol of phosphate per minute from inorganic pyrophosphate at 75°C and pH 8.5.

Procedure

Usage Instructions

The Thermostable Pyrophosphatase can reduce the inhibitory effects of accumulated PPi in polymerase chain reactions (PCRs) (5-7). As a starting point for PCR, add 0.01-0.1 U/µL of Thermostable Pyrophosphatase to identify the optimal concentration. Notes: Thermostable Pyrophosphatase catalysis is Mg 2+- dependent. Therefore, it is important to have Mg 2+ in the reaction buffer. It is compatible with most PCR buffers that contain Mg2 + .

Quality Control

Unit activity

The assay is based on that described by Taussky and Shorr (4). Briefly, enzyme dilutions are added to 30 mM Tris HCl pH 8.5, 1.5 mM MgCl 2 and 1.5 mM sodium pyrophosphate. After a 10-minute incubation at 75°C, the product formed, 2-orthophosphate, is reacted with ammonium molybdate to form phosphomolybdic acid. The phosphomolybdic acid is then reduced by ferrous sulfate under weak acidic conditions to form a blue color, the absorbance of which is measured at 660 nm. The amount of product formed is extrapolated from a phosphate standard curve generated from the ammonium molybdate/ferrous sulfate reaction. Protein concentration (OD 280 ) is determined by OD 280 absorbance. Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample. Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded endonuclease is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C. E. coli 16S rDNA contamination is evaluated using 5 µL replicate samples of enzyme solution denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Applications

- Production of orthophosphate
- Avoiding inhibition in RNA synthesis and modification reactions
- Avoiding inhibition in PCR reactions

- Leppänen, V.M. et al. (1999) Protein Sci., 8(6):1218-31.
- Hansen, T. et al. (1999) Arch. Biochem. Biophys., 363(1):135-47.
- Meyer, W. et al. (1995) Arch. Biochem. Biophys., 319(1):149-56.
- Taussky, H.H. and Shorr, E. (1953) J. Biol. Chem., Jun;202(2):675-85.
- Moldes, C. et al. (2004) Appl. Environ. Microbiol., 70(8): 4642-4647.
- Dignam, J.D. and Deutscher, M.P. (1979) Biochemistry, 18(14):3165-70.
- Gibson, N.J. et al. (1997) Anal. Biochem., 254(1):18-22.

This product is available for molecular biology applications such as:

References