Datasheet for T7 RNA Polymerase

Applications: 1X RNAPol Reaction Buffer: Incubate at 37°C for 1 hour. For shorter (< 300 nt) • Radiolabeled RNA probes 40 mM Tris-HCl transcripts incubate at 37°C for 2–16 hours. T7 RNA Polymerase 6 mM MgCl • Non-isotopic RNA labeling 2 2 mM spermidine Notes on use: • Preparation of RNA vaccines 1 mM dithiothreitol For radio labeled high specific activity RNA 1-800-632-7799 • Guide RNA for gene targeting pH 7.9 @ 25°C probes, the concentration of the radioactive [email protected] nucleotide should be limited to 6 μM. www.neb.com • mRNA for in vitro translation and micro injection Unit Definition: One unit is defined as the amount of M0251S 013150717071 • RNA structure, processing and catalysis studies enzyme required to incorporate 1 nmol ATP into an To protect RNA against ribonuclease, RNase inhibitor (NEB #M0314 or #M0307) should be • RNA amplification acid-insoluble material in 1 hour at 37°C. added to a final concentration of 1 U/μl. M0251S • Anti-sense RNA for gene expression experiment Unit Assay Conditions: 1X RNAPol Reaction Buffer, T7 RNA Polymerase is extremely sensitive to salt supplemented with 0.5 mM each ATP, UTP, GTP, CTP 5,000 units 50,000 U/ml Lot: 0131507 Supplied in: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9), inhibition. The overall salt concentration should and 1 µg T7 DNA in 50 µl. not exceed 50 mM. RECOMBINANT Store at –20°C Exp: 7/17 1 mM EDTA, 20 mM 2-mercaptoethanol, 0.1% Triton X-100 and 50% glycerol. Description: Bacteriophage T7 RNA Polymerase is Protocol for Standard RNA Synthesis: Assemble the Quality Control Assays a DNA-dependent RNA polymerase that is highly reaction at room temperature in the following order. Reagents Supplied with Enzyme: Endonuclease Activity (Nicking): A 50 μl reaction specific for the T7 phage promoters. The 99 KD 10X RNAPol Reaction Buffer. COMPONENTS AMOUNT CONCENTRATION in RNAPol Reaction Buffer containing 1 μg of enzyme catalyzes in vitro RNA synthesis from a H2O X µl supercoiled φX174 DNA and a minimum of cloned DNA sequence under the T7 promoters. Reaction Conditions: 1X RNAPol Reaction Buffer, 10X Reaction Buffer 2 µl 150 units of T7 RNA Polymerase incubated for RNA produced using the T7 RNA Polymerase is supplemented with 0.5 mM each ATP, UTP, GTP, CTP NTP X µl 0.5 mM each 4 hours at 37°C results in < 10% conversion to suitable for many applications in research and and DNA template containing the T7 RNA Polymerase Template DNA X µl 0.2–1 µg the nicked form as determined by agarose gel biotechnology. promoter. Incubate at 37°C. RNase Inhibitor (optional) 0.5 µl 1 U/µl final electrophoresis. Fresh DTT (optional) X µl 5 mM final Source: Isolated from E. coli BL21 carrying the T7 RNA Pol 2 µl plasmid pAR1219 which contains T7 gene I. Total Volume 20 µl (see other side)

CERTIFICATE OF ANALYSIS

CERTIFICATE OF ANALYSIS Exonuclease Activity (Radioactivity Release): Protein Purity Assay (SDS-PAGE): T7 RNA Low Range ssRNA Ladder A 50 μl reaction in RNAPol Reaction Buffer Polymerase is ≥ 95% pure as determined by SDS- #N0364S 25 µg containing 1 μg of a mixture of single and double- PAGE analysis using Coomassie Blue detection. ISO 9001 ISO 14001 ISO 13485 ssRNA Ladder Registered Registered Registered stranded [³H] E. coli DNA and a minimum of Quality Environmental Medical Devices #N0362S 25 µg Management Management 150 units of T7 RNA Polymerase incubated for RNase Activity (Extended Digestion): A 10 μl NEW ENGLAND BIOLABS® is a registered trademark of New England 4 hours at 37°C releases < 0.1% of the total reaction in RNAPol Reaction Buffer containing RNase Contamination Assay Kit Biolabs, Inc. radioactivity. 40 ng of a 300 base single-stranded RNA and a #E3320S 50 reactions This product is intended for research purposes only. This product is not minimum of 50 units of T7 RNA Polymerase is intended to be used for therapeutic or diagnostic purposes in humans Vaccinia Capping System or animals. Non-Specific DNase Activity (16 Hour): A 50 μl incubated at 37°C. After incubation for 4 hours, #M2080S 400 units reaction in RNAPol Reaction Buffer containing > 90% of the substrate RNA remains intact 1 μg of Lambda DNA and a minimum of 250 units as determined by gel electrophoresis using mRNA Cap 2´-O-Methyltransferase of T7 RNA Polymerase incubated for 16 hours at fluorescent detection. #M0366S 2,000 units 37°C results in a DNA pattern free of detectable E. coli Poly(A) Polymerase nuclease degradation as determined by agarose gel Companion Products: #M0276S 100 units electrophoresis. RNA Loading Dye (2X) #M0276L 500 units #B0363S 4 x 1 ml Promoter Specificity: A 50 μl reaction in RNAPol Ribonucleotide Solution Mix Reaction Buffer in the presence of 2 mM NTPs RNase Inhibitor, Human Placenta #N0446S 10 μmol of each containing 1 μg of Lambda DNA as a template and #M0307S 2,000 units #N0446L 50 µmol of each a minimum of 200 units of T7 RNA Polymerase #M0307L 10,000 units Ribonucleotide Solution Set incubated for 1 hour at 37°C results in < 1.5% of the RNase Inhibitor, Murine #N0450S 10 µmol each amount of product incorporated as compared to a #M0314S 3,000 units #N0450L 50 µmol each control reaction using T7 DNA as a template. #M0314L 15,000 units SP6 RNA Polymerase Pyrophosphatase, Inorganic (E. coli) #M0207S 2,000 units #M0361S 10 units #M0207L 10,000 units Page 2 (M0251) #M0361L 50 units

Exonuclease Activity (Radioactivity Release): Protein Purity Assay (SDS-PAGE): T7 RNA Low Range ssRNA Ladder A 50 μl reaction in RNAPol Reaction Buffer Polymerase is ≥ 95% pure as determined by SDS- #N0364S 25 µg containing 1 μg of a mixture of single and double- PAGE analysis using Coomassie Blue detection. ISO 9001 ISO 14001 ISO 13485 ssRNA Ladder Registered Registered Registered stranded [³H] E. coli DNA and a minimum of Quality Environmental Medical Devices #N0362S 25 µg Management Management 150 units of T7 RNA Polymerase incubated for RNase Activity (Extended Digestion): A 10 μl NEW ENGLAND BIOLABS® is a registered trademark of New England 4 hours at 37°C releases < 0.1% of the total reaction in RNAPol Reaction Buffer containing RNase Contamination Assay Kit Biolabs, Inc. radioactivity. 40 ng of a 300 base single-stranded RNA and a #E3320S 50 reactions This product is intended for research purposes only. This product is not minimum of 50 units of T7 RNA Polymerase is intended to be used for therapeutic or diagnostic purposes in humans Vaccinia Capping System or animals. Non-Specific DNase Activity (16 Hour): A 50 μl incubated at 37°C. After incubation for 4 hours, #M2080S 400 units reaction in RNAPol Reaction Buffer containing > 90% of the substrate RNA remains intact 1 μg of Lambda DNA and a minimum of 250 units as determined by gel electrophoresis using mRNA Cap 2´-O-Methyltransferase of T7 RNA Polymerase incubated for 16 hours at fluorescent detection. #M0366S 2,000 units 37°C results in a DNA pattern free of detectable E. coli Poly(A) Polymerase nuclease degradation as determined by agarose gel Companion Products: #M0276S 100 units electrophoresis. RNA Loading Dye (2X) #M0276L 500 units #B0363S 4 x 1 ml Promoter Specificity: A 50 μl reaction in RNAPol Ribonucleotide Solution Mix Reaction Buffer in the presence of 2 mM NTPs RNase Inhibitor, Human Placenta #N0446S 10 μmol of each containing 1 μg of Lambda DNA as a template and #M0307S 2,000 units #N0446L 50 µmol of each a minimum of 200 units of T7 RNA Polymerase #M0307L 10,000 units Ribonucleotide Solution Set incubated for 1 hour at 37°C results in < 1.5% of the RNase Inhibitor, Murine #N0450S 10 µmol each amount of product incorporated as compared to a #M0314S 3,000 units #N0450L 50 µmol each control reaction using T7 DNA as a template. #M0314L 15,000 units SP6 RNA Polymerase Pyrophosphatase, Inorganic (E. coli) #M0207S 2,000 units #M0361S 10 units #M0207L 10,000 units Page 2 (M0251) #M0361L 50 units