Poly(A) Polymerase, 500 U (for mRNA synthesis related enzymes)

Product Information

Product Introduction

Poly(A) Polymerase, also known as Poly(A) tailing enzyme, is derived from Escherichia coli. It catalyzes the addition of AMP converted from ATP to the 3' end of single-stranded RNA in a template-independent manner to form a Poly(A) tail. Poly(A) RNA Polymerase has a high tailing efficiency and can add 20 to 200 A bases to the 3' end of RNA. Main uses: RNA end labeling, in vitro mRNA synthesis, etc.

Source: Derived from Escherichia coli and expressed by recombinant E. coli.

Enzyme activity definition: Under the conditions of 37°C and pH 8.0, the amount of enzyme required to incorporate 1 nmol AMP into the RNA terminus within 10 minutes is defined as 1 unit of enzyme activity.

Purity and concentration: Purity detected by SDS-PAGE>95%; residual endogenous nucleic acid <1 pg/μL (qPCR detection); 5 U/μL.

Inactivation or inhibition: Inactivation by adding EDTA to a concentration of 10 nM or heating at 65°C for 20 min.

Enzyme storage buffer: 20 mM Tris-HCl, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100, 50% Glycerol, pH 7.5.

10хPAP Reaction buffer：500 mM Tris-HCl，2.5 mM NaCl，100 mM MgCl₂，pH 8.0。

Figure 1. Effect of adding poly(A) tail to 300 nt single-stranded RNA. 20 μL reaction system, 0.5 μg single-stranded RNA (300 nt), different amounts of Poly(A) RNA Polymerase were added, incubated at 37 for 30 min, and terminated by incubation at 65 for 20 min.

Storage and transportation

Transported with wet ice; stored at -20, valid for 12 months.

composition

Procedure

1. Configure the reaction system according to the following table:

2. Incubate the reaction system at 37°C for 30 min. Add EDTA to a final concentration of 10 mM or heat at 65°C for 20 min to terminate the reaction. (Note: RNA used for tailing reaction should be properly purified.)

Precautions

1. When it comes to RNA operations, they must be performed strictly in accordance with RNA operation specifications to avoid RNase contamination. Relevant reagents and consumables must be treated with DEPC to remove RNase or ensure that they are RNase free.

2. The enzyme can also be used for the reaction using M-MuLV reverse transcriptase reaction buffer, which requires divalent cations such as Mg ^{2+ to be active.}

3. The length of the A tail added to RNA is affected by factors such as the amount of enzyme, ATP concentration and reaction time. Different experiments require different amounts of A. The length of the added A can be adjusted by reducing the reaction time. The enzyme can add about 30 A bases when reacting at 37°C for 30 min, and about 100 A bases in 1 h.

4. For your safety and health, please wear a lab coat and disposable gloves when operating.

The product is for scientific research purposes only and not for clinical diagnosis!

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