Pyrophosphatase Inorganic (yeast)

Pyrophosphatase Inorganic (yeast) is derived from a recombinant E. coli strain carrying Saccharomyces cerevisiae ppa gene. The enzyme catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate. P207–4 + H20 → 2HP04–2 Pyrophosphatase Inorganic (yeast) could hydrolyze the inorganic pyrophosphate generated by nucleic acid amplification, in vitro transcription and other reactions, removing the inhibition of the inorganic pyrophosphate generated on the reaction system. The removal of pyrophosphate can shift the reaction equilibrium to the end of product formation, which is beneficial to increase the yield of synthetic product. RNA and DNA synthesis are examples of reactions that can be pulled far in the synthesis direction by the action of inorganic pyrophosphatase.

Storage

Store at -20 ℃, valid for 2 years（ Avoid repeated freeze-thaw cycles）

Storage Buffer

20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol，50% glycerol。

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. 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.

Molecular Weight

The molecular weight of Pyrophosphatase, Inorganic (yeast) is 71 kDa.

Quality Control

Exonuclease: 0.1 U of Pyrophosphatase, Inorganic (yeast) with 1 μg λ-Hind III digest DNA at 37 ℃ for 16 hours yields no degradation as determined by agarose gel electrophoresis.

Endonuclease: 0.1 U of Pyrophosphatase, Inorganic (yeast) with 1 μg λDNA at 37 ℃ for 16 hours yields no degradation as determined by agarose gel electrophoresis.

Nickase: 0.1 U of Pyrophosphatase, Inorganic (yeast) with 1 μg pBR322 at 37 ℃ for 16 hours yields no degradation as determined by agarose gel electrophoresis.

RNase: 0.1 U of Pyrophosphatase, Inorganic (yeast) with 1.6 μg MS2 RNA for 4 hours at 37 ℃ yields no degradation as determined by agarose gel electrophoresis.

E. coli DNA: 0.1 U of Pyrophosphatase, Inorganic (yeast) is screened for the presence of E.coli genomic DNA using TaqMan qPCR with primers specific for the E. coli 16S rRNA locus. The E. coli genomic DNA contamination is ≤ 0.1 pg/ 0.1U.

Bacterial Endotoxin: LAL-test, according to Chinese Pharmacopoeia IV 2020 edition, gel limit test method, general rule (1143). Bacterial endotoxin content should be ≤10 EU/mg.

Note on use

1. This product is active in a variety of reaction buffers, usually this product can be directly connected in HDA and LAMP amplification experiments.

2. It is recommended to optimize the dosage of the enzyme in different experiments, and the concentration is usually 0.05-1 U/mL. 4 U/mL is widely adopted for in vitro transcription system.

3. Pyrophosphatase, Inorganic (yeast) is active from 16 to 37°C while the optimum reaction temperature is 25°C.

4. For your safety, please wear a lab coat and disposable gloves during operation.

Enhancing yields of RNA in transcription reactions