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E. coli Poly(A) Polymerase
Recombinant Source37
Catalog # Size Concentration Price Qty  
M0276L 500 units 5,000 units/ml $252.00
M0276S 100 units 5,000 units/ml $63.00
Prices are in US dollars and valid only for US orders.
Download:MSDS PDF


  • For the 3' labeling of RNA
  • For preparing a priming site for cDNA synthesis using oligo-dT
  • Enhances translation of RNA transferred into eukaryotic cells
Description:
Poly(A) Polymerase catalyzes the template independent addition of AMP from ATP to the 3´ end of RNA.

Source:
An E. coli strain that carries the cloned Poly(A) Polymerase gene from E. coli (1).

Applications:
  • Labeling of RNA with ATP or cordycepin
  • Poly(A) tailing of RNA for cloning or affinity purification
  • Enhances translation of RNA transferred into eukaryotic cells.
Reagents Supplied:
Poly(A) PolymeraseReaction Buffer (10X)
ATP (10 mM)


Reaction & Storage Conditions


Reaction Conditions:
1X Poly(A) PolymeraseReaction Buffer
Supplemented with 1 mM ATP
Incubate at 37°C.

1X Poly(A) PolymeraseReaction Buffer:
50 mM Tris-HCl
250 mM NaCl
10 mM MgCl2
pH 7.9 @ 25°C

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. 

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.

Concentration:
5,000 units/ml

Storage Conditions:
20 mM Tris-HCl
300 mM NaCl
1 mM DTT
1 mM EDTA
50% Glycerol
0.1% Triton X-100


Storage Temperature:
-20°C


Notes


General notes:
  1. rATP is not included in the buffer supplied.

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:
Poly(A) Polymerase contains no detectable DNAses, RNAses and phosphatases. The purified protein contains no detectable DNA or RNA as determined by ethidium staining of an agarose gel.

RNase Assay:
Incubation of a 10 µl reaction containing 5 units of E. coli Poly(A) Polymerase with 40 ng of RNA transcript for 2 hours at 37°C resulted in no detectable degradation of the RNA as determined by gel electrophoresis.

DNA Exonuclease Activity:
Incubation of a 50 µl reaction containing 10 units of Poly(A) Polymerase with 1 µg of a mixture of single and double-stranded 3H E.coli DNA (200,000 cpm/µg) for 3 hours at 37°C released < 0.1% of the total radioactivity.

DNA Endonuclease Activity:
Incubation of a 10 µl reaction containing 5 units of E. coli Poly(A) Polymerase with 40 ng of RNA transcript for 2 hours at 37°C resulted in no detectable degradation of the RNA as determined by gel electrophoresis.


References


  1. Cao, G.J. and Sarkar, N. (1992) Proc. Natl. Acad. Sci. USA, 89, 10380-10384.

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