Poly(A) Polymerase catalyzes the template independent addition of AMP from ATP to the 3´ end of RNA.
Figure 1. Analysis of capped and polyadenylated RNA
A. Agilent Bioanalyzer® analysis of capped and polyadenylated RNA. Longer tails are produced by increasing the enzyme concentration in the reaction. Calculated A-tail lengths are indicated over each lane. Lanes: L: size marker,1: No poly-A tail, 2: 5 units, 3 :15 units, 4 : 25 units of E. coli Poly(A) Polymerase. B. Effect of enzymatic A-tailing on the luciferase reporter activity of CLuc mRNA
Product Source
An E. coli strain that carries the cloned Poly(A) Polymerase gene from E. coli (1).
Reagents Supplied
The following reagents are supplied with this product:
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Advantages and Features
Application Features
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Labeling of RNA with ATP or cordycepin
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Poly(A) tailing of RNA for cloning or affinity purification
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Enhances translation of RNA transferred into eukaryotic cells.
Properties & Usage
Unit Definition
One unit is defined as the amount of enzyme that will incorporate 1 nmol of AMP into RNA in a 20 µl volume in 10 minutes at 37°C.
Reaction Conditions
1X Poly(A) Polymerase Reaction Buffer
Incubate at 37°C
1X Poly(A) Polymerase Reaction Buffer
50 mM Tris-HCl
250 mM NaCl
10 mM MgCl2
(pH 8.1 @ 25°C)
Storage Buffer
20 mM Tris-HCl
300 mM NaCl
1 mM DTT
1 mM EDTA
50% Glycerol
0.1% (w/v) Triton® X-100
pH 7.5 @ 25°C
Unit Assay Conditions
1X Poly(A) Polymerase Reaction Buffer, 1 mM rATP and 500 ng 5´ FAM labeled poly A 20-mer RNA in a 20 µl reaction. After incubation at 37°C for 10 minutes the length of the poly(A) addition is determined either by gel electrophoresis or with an automated capillary DNA sequencer. In this assay 5 units of enzyme add approximatley 60 to 80 adenosines to the RNA primer. In these conditons 20 units of enzyme will deplete the rATP.
Product Notes
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rATP is not included in the buffer supplied.
References
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Cao, G.J. and Sarkar, N. (1992). Proc.Natl. Acad. Sci. USA. 89, 10380-10384.
Protocols
Datacards
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.)
Selection Charts
Selection Charts
Quality Control Assays
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The following link is to a PDF Safety Data Sheet (SDS) that applies to this product to help you use it safely.
Datacards
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.)
Legal and Disclaimers
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please email us at busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science - we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.