 |
|
 |
|
|
| Home >
Products >
DNA Modifying Enzymes and Cloning >
Phosphatases and Sulfurylases >
Pyrophosphatase, Inorganic (yeast) |
 | | | | Pyrophosphatase, Inorganic (yeast) | | | Developed and produced by BioHelix Corp., a NEB-affiliated company. | |
| |
|
|
|
 |
|
Prices are in US dollars and valid only for US orders.
|
- Isolated from a recombinant source
Description:
Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate.
P2O7-4 + H2O -> 2HPO4-2
A variety of metabolic reactions generate inorganic pyrophosphate as a reaction byproduct. Such reactions are rendered irreversible when the pyrophosphate is degraded by pyrophosphatase (1). RNA and DNA synthesis are examples of reactions that can be pulled far in the synthesis direction by the action of inorganic pyrophosphatase.
Source:
PPase is prepared from an E. coli strain containing a genetic fusion of the Saccharomyces cerevisiae ppa gene and the gene coding for Mycobacterium xenopi GyrA intein. The final product has been cleaved and purified from the intein fusion partner. Developed and produced by the Biohelix Corporation, a New England Biolabs-affiliated company.
Applications:- Enhancing yields of RNA in transcription reactions (2)
Enzyme Properties
Molecular Weight:
Theoretical: 71 kDa
Reaction & Storage Conditions
Unit Definition:
One unit is the amount of enzyme that will generate 1 µmol of phosphate per minute from inorganic pyrophosphate under standard reaction conditions (a 10 minute reaction at 25°C in 100 mM Tris-HCl, [pH 7.2], 2 mM MgCl2 and 2 mM PPi in a reaction volume of 0.5 ml).
Concentration:
100 units/ml
Storage Conditions:
20 mM Tris-HCl
100 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 8.0 @ 25°C
Storage Temperature:
-20°C
FAQs
- Can alternate metals be used as activators for Yeast Inorganic Pyrophosphatase?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Free of endonuclease, exonuclease and RNase activities.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 2 units of Pyrophosphatase, Inorganic (yeast) with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 2 units of Pyrophosphatase, Inorganic (yeast) with 1 μg of
ΦX174 DNA for 4 hours at 25ºC resulted
in < 1% conversion to RFII as determined by agarose gel electrophoresis.
RNase Assay:
Incubation of a 50 μl reaction containing 2 units of Pyrophosphatase, Inorganic (yeast) with 1
μg of MS2 RNA for 1 hour at 25ºC
resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis.
DNase Activity:
Incubation of a 50 μl reaction containing 2 units of Pyrophosphatase, with 1 μg of λ DNA (HindIII digest) for 16 hours at 37°C resulted the same pattern of DNA bands as a reaction without enzyme as determined by agarose gel electrophoresis.
Alkaline Phosphatase Activity:
This colorimetric assay tests for the presence of alkaline phosphatase which removes 5´ phosphates from DNA, RNA, rNTPs and dNTPs. Phosphatase contamination is revealed if p-nitrophenylphosphate is hydrolyzed to p-nitrophenol (yellow color). The PPase is incubated in a reaction buffer (1 ml) of 1 M diethanolamine-HCl (pH 9.8), 0.5 mM MgCl2 and 10 mM p-nitrophenylphosphate at 37°C. Conversion of p-nitrophenylphosphate to p-nitrophenol is measured spectrophotometrically at A405 after 60 minutes. One unit of alkaline phosphatase is defined as the amount of enzyme that hydrolyzes 1 µmole of p-nitrophenylphosphate to p-nitrophenol in 1 minute. When 2 units of PPase are incubated under the above conditions < 0.0001 unit of alkaline phosphatase activity is revealed.
dNTPase Activity:
dNTPase contamination is measured as the removal of β or γ phosphates from dATP, dCTP, dGTP, or dTTP using the AAM assay (1) for inorganic phosphate. The PPase is incubated in a volume of 0.5 ml @ 25°C for 1 hour in CircumVent™ Sequencing Buffer with a mixture of dNTPs, each at 200 µM. Incubation under these conditions with 5 units of PPase liberated < 0.05 µmole of inoganic phosphate from dNTPs.
References
- Kornberg, A. (1980) DNA Replication, 54-55, 94. W.H. Freeman and Company..
- Cunningham, P.R. and Ofengand, J. (1990) Biotechniques, 9, 713-714.
- Cooperman, B.S. (1982) Methods Enzymol., 87 (pt. C), 526-548.
|
|
|
 |
|
|
