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E. coli Poly(A) Polymerase |
 | | | | E. coli Poly(A) Polymerase | | | |
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Prices are in US dollars and valid only for US orders.
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- 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)
Reaction & Storage Conditions
Reaction Conditions:
1X Poly(A) PolymeraseReaction Buffer
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:
25 mM Tris-HCl
200 mM NaCl
0.1 mM DTT
0.1 mM EDTA
50% Glycerol
0.1% Triton X-100
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- 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 50 µl reaction containing 10 units of Poly(A) Polymerase with 3 µg of ssRNA ladder for 3 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 50 µl reaction containing 10 units of Poly(A) Polymerase with 1 µg of φX174 RF I DNA for 3 hours at 37°C resulted in < 10% conversion to RFII as determined by agarose gel electrophoresis.
References
- Cao, G.J. and Sarkar, N. (1992) Proc. Natl. Acad. Sci. USA, 89, 10380-10384.
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