Poly(A) Polymerase catalyzes the addition of AMP from ATP to the 3' hydroxyl of RNA. The reaction requires Mg2+ and is template independent.
Source of Protein
The gene encoding E. coli Poly(A) Polymerase expressed from a plasmid in E. coli.
25 mM Tris-HCl
500 mM NaCl
0.1 mM DTT
0.1 mM EDTA
pH 8.0 @ 25°C
B7460 (10X Poly(A) Polymerase Reaction Buffer)
N2070-10 (10 mM ATP Solution)
10X Poly(A) Polymerase Reaction Buffer (B7460):
500 mM Tris-HCl
2.5 M NaCl
100 mM MgCl2
pH 7.9 @ 25°C
1 unit is defined as the amount of enzyme that will incorporate 1 nmol of ATP into acid-insoluble material in 10 minutes at 37°C.
Single-Stranded Exonuclease Activity
A 50 µL reaction containing 10,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 5% release of TCA-soluble counts.
Double-Stranded Exonuclease Activity
A 50 µL reaction containing 5,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 1% release of TCA-soluble counts.
Double-Stranded Endonuclease Activity
A 50 µL reaction containing 0.5 µg of pBR322 DNA and 10 µÁL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.
E.coli 16S rDNA Contamination Test
Replicate 5 µL samples of enzyme solution were denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.
Non-Specific RNAse Assay
Replicate 10 µL samples were screened for non-specific RNAse contamination using the RNAse Alert kit, (Integrated DNA Technologies), following the manufacturer's guidelines.
||-15 to -25°C
||200 U <5.0% released
||200 U <1.0% released
||200 U = no conversion
|E.coli DNA Contamination
||100 U <10 copies
||200 U = no detectable non-specific RNAse
* For a detailed summary of assay conditions and data,
refer to the Quality Controls Analysis section below
Quality Control Analysis:
Unit Characterization Assay
SSpecific activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer (50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl2, pH 7.9 @ 25°C, and added to 50 µL reactions containing a 15-mer RNA Oligo, 1X reaction buffer, 1 mM ATP, 2.5 mM MnCl2 and 3H-ATP. Reactions were incubated 10 minutes at 37°C, plunged on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).
Protein Concentration (OD280) Measurement
A 2.0 µL sample of enzyme was analyzed at OD280 using a Nanodrop ND-2000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209), and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 52,060 and molecular weight of 53,870 Daltons.
SDS-Page (Physical Purity Assessment)
2.0 µL of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µL of a 1:100 dilution of the sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 95% purity of the concentrated sample.