Description
Poly(A) Polymerase attaches ATP (catalyzed from AMP) to the 3ʹ hydroxyl of RNA, which is useful for making poly(A)-tailed RNA. The enzyme is supplied in 25 mM Tris-HCl, 500 mM NaCl, 1 mM MgCl 2, 0.1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 8.0 at 25°C. The reaction buffer for Poly(A) Polymerase at 10x concentration includes 500 mM Tris-HCl, 2.5 M NaCl, and 100 mM MgCl 2, with a pH of 7.9 at 25°C.
Poly(A) Polymerase catalyzes the addition of AMP from ATP to the 3ʹ hydroxyl of RNA. The reaction requires Mg 2+ and is template independent. Storage temperature: –25°C to –15°C Molecular weight: 53,870 Daltons Test Units tested Specification Purity >95% Specific activity >20,000 U/mg Single-stranded exonuclease 200 U <5% released Double-stranded exonuclease 200 U <1% released Double-stranded endonuclease 200 U No conversion E. coli DNA contamination 100 U <10 copies RNase contamination 200 U No detectable non-specific RNase
The protein is produced by an E. coli strain expressing the Poly(A) Polymerase gene from a plasmid. One 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.
Usage Instructions 1. Set up the following reaction mixture in a total volume of 20 µL in the order listed: 1-10 µg purified RNA in 15 µL of nuclease-free water 2 µL 10x Poly(A) Polymerase Reaction Buffer (B7460) 2 µL 10mM ATP (N2070-10) 1 µL Poly(A) Polymerase (P7460L) 2. Incubate the reaction mixture at 37°C for 30 minutes. 3. Stop the reaction by adding EDTA (final concentration of 10mM) or proceed to the cleanup step. Quality Control Specific activity was measured using a twofold serial dilution method. Dilutions of the enzyme were made in 1x reaction buffer (50 mM Tris-HCl, 250 mM NaCl, 10 mM MgCl 2, pH 7.9 at 25°C, and added to 50 µL reactions containing a 15-mer RNA oligo, 1x reaction buffer, 1 mM ATP, 2.5 mM MnCl 2 and 3H-ATP. Reactions were incubated for 10 minutes at 37°C, placed on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26). Protein concentration is determined by OD 280 absorbance. Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the band's mass corresponding to the protein of interest in the diluted sample. Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded exonuclease activity is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded endonuclease activity is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C. E. coli contamination is evaluated using 5 µL replicate samples of enzyme solution that are 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. Non-specific RNAse contamination is assessed using the RNAse Alert kit (Integrated DNA Technologies), following the manufacturer’s guidelines.
