The E. coli Poly(A) tailing enzyme produced by our company, also known as E. coli Poly(A) polymerase, E. coli PAP, PAP enzyme, Poly(A) tailing enzyme or PolyA tailing enzyme, catalyzes the template-independent addition of AMP derived from ATP to the 3'-end of single-stranded RNA to form a Poly(A) tail. E. coli Poly(A) polymerase can use various single-stranded RNAs as substrates. However, double-stranded RNA and excessively short oligonucleotides are not recommended as substrates for this reaction, and DNA cannot be used as a substrate. It has been experimentally verified that 15nt synthetic RNA can fully serve as a substrate for E. coli Poly(A) polymerase to catalyze the formation of a Poly(A) tail in the presence of ATP. The Poly(A) tailing reaction catalyzed by E. coli Poly(A) polymerase can only use ATP, not ADP or dATP. In addition, when using CTP or UTP, the incorporation efficiency is less than 5% of that with ATP; while GTP cannot be added to the 3'-end of RNA at all.
| Source | Recombinantly expressed in E. coli
| | Appearance | Sterile liquid
| | Storage Buffer | 20mM Tris-HCl (pH7.5, 25℃), 500mM NaCl, 1mM DTT, 1mM EDTA, 50% (v/v) Glycerol, 0.1% (w/v) Triton X-100 | | Enzyme Concentration | 5U/μL | | Purity | Free of DNase, RNase and phosphatase
| | Activity Definition | One unit is defined as the amount of enzyme that will incorporate 1nmol of AMP into RNA in a 20μl volume in 10 minutes at 37℃ |
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Components List
| P1522669 | Components | Appearance | 100U | 5×100U | Storage | | P1522669A | E.coli Poly(A) Polymerase (5U/µl) | Liquid | 20µl | 5×20µl | -20℃. Avoid freeze/thaw cycle. | | P1522669B | 10× PAP Reaction Buffer | Liquid | 50µl | 5×50µl | -20℃. Avoid freeze/thaw cycle. | | P1522669C | 10mM ATP | Liquid | 50µl | 5×50µl | -20℃. Avoid freeze/thaw cycle. |
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Product Applications
Labeling RNA with radioisotope-labeled ATP or cordycepin; Adding Poly(A) tails to RNA for post-reverse transcription cloning or affinity purification; Increasing mRNA stability to improve translation efficiency of RNA transfected into eukaryotic cells.
Product Advantages
This product catalyzes the template-independent addition of AMP derived from ATP to the 3'-end of single-stranded RNA to form a Poly(A) tail.
Instructions for Use
1. Set up the reaction system on ice according to the table below. The amount of RNA substrate for Poly(A) tailing in the 20μl reaction system can be up to 10μg, and the reaction system can be scaled up proportionally according to experimental needs. In the Poly(A) tailing reaction, the tail length depends on the molar concentration of the RNA 3'-OH end, reaction time, enzyme amount, and ATP concentration. The tail length can be adjusted by changing one or more of these factors. Using the recommended reaction system below and incubating at 37℃ for 30min, the Poly(A) tail length can exceed 100 bases.
| Reagent | Volume | Final Concentration |
|---|
| DEPC-treated Water | (14.5-x)µl | - | | 10× PAP Reaction Buffer | 2µl | 1X | | 10mM ATP | 2µl | 1mM | | RNase Inhibitor (40U/µl) | 0.5µl | 1U/µl (Optional) | | RNA | xµl | 0.05-0.5µg/µl | | E.coli Poly(A) Polymerase (5U/µl) | 1µl | 0.25U/µl | | Total Volume | 20µl | - |
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Note 1: Since RNA handling is involved, strictly follow RNA operation specifications to avoid RNase contamination. Relevant reagents and consumables should be treated with DEPC to remove RNase or be confirmed as RNase-free.
Note 2: RNA used for the tailing reaction should be properly purified before use, dissolved in nuclease-free water, and the solution should contain no EDTA or salts.
Note 3: If strict RNase-free conditions are difficult to ensure, it is recommended to add 0.5µl RNase Inhibitor to the above reaction system to improve RNA stability in the solution.
2. Tailing reaction: Incubate at 37℃ for 30min.
3. Reaction termination: Stop the reaction by adding EDTA to a final concentration of 10mM, or heating at 65℃ for 20min.
Storage Conditions
Store at -20℃, valid for at least 2 years. Transport at ≤0℃.
Precautions
(1) The 10× PAP Reaction Buffer does not contain ATP.
(2) The amount of E. coli Poly(A) Polymerase mainly affects the length of the target product. The enzyme amount can be adjusted according to the desired product length. To obtain longer products, appropriately increase the enzyme amount or extend the reaction time.
(3) This product is for scientific research use only by professionals. It shall not be used for clinical diagnosis or treatment, food or pharmaceutical applications, nor stored in ordinary residential premises.
(4) For your safety and health, wear a lab coat and disposable gloves when handling.
