Datasheet for T7 Exonuclease (M0263; Lot 0031212)
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Source: Purified from an E. coli strain containing a Unit Assay Conditions: 50 mM potassium Physical Purity: Purified to > 95% homogeneity TYB12 intein fusion acetate, 20 mM Tris-acetate, 10 mM magnesium as determined by SDS-PAGE analysis using T7 Exonuclease acetate, 1mM dithiothreitol (pH 7.9) and 0.15 mM Coomassie Blue detection. Supplied in: 10 mM Tris-HCl (pH 8.0), sonicated duplex [3H] DNA. 0.1 mM EDTA, 5 mM DTT and 50% glycerol. RNase Activity (Extended Digestion): A 10 µl Quality Control Assays 1-800-632-7799 reaction in NEBuffer 4 containing 40 ng of [email protected] Reagents Supplied with Enzyme: Single Stranded Deoxyribonuclease Activity flourescein labeled RNA transcript and 10 units www.neb.com 10X NEBuffer 4. (FAM Labeled Oligo): A 50 µl reaction in of T7 Exonuclease incubated at 37°C. After M0263S 003121214121 NEBuffer 4 containing a 20 nM solution of a incubation for 4 hours, > 90% of the substrate Reaction Conditions: fluorescent internal labeled oligonucleotide and a RNA remains intact as determined by gel minimum of 50 units of T7 Exonuclease incubated 1X NEBuffer 4. electrophoresis using fluorescence detection. M0263S Incubate at 25°C. for 16 hours at 37°C yields < 5% degradation as determined by capillary electrophoresis. 1,000 units 10,000 U/ml Lot: 0031212 Heat Inactivation: No 1X NEBuffer 4: RECOMBINANT Store at –20°C Exp: 12/14 50 mM potassium acetate Endonuclease Activity: Incubation of a 50 µl References: reaction containing 100 units of T7 Exonuclease Description: T7 Exonuclease acts in the 5´ to 20 mM Tris-acetate 1. Kerr, C. and Sadowski, P.D. (1972) J. Biol. with 1 µg of φX174 RF I DNA for 4 hours at 25°C 3´ direction, catalyzing the removal of 5´ mono- 10 mM magnesium acetate Chem. 247, 305–318. resulted in < 10% loss in supercoiled DNA as nucleotides from duplex DNA. T7 Exonuclease 1 mM DTT 2. Shinozaki, K. and Okazaki, T. (1978) Nucleic determined by agarose gel electrophoresis. is able to initiate nucleotide removal from the pH 7.9 @ 25°C Acids Res. 5, 4245–4261. 5´ termini or at gaps and nicks of double-stranded Functional Assay (RNase, RNA/DNA Hybrid): DNA (1). It will degrade both 5´ phosphorylated Unit Definition: One unit is defined as the amount Incubation of 10 units of T7 Exonuclease with or 5´ dephosphorylated DNA. It has been also of enzyme to produce 1 nmol of acid soluble 20 nmol [3H]poly(A).poly(dT) hybrid polymer ISO 9001 ISO 14001 ISO 13485 reported to degrade RNA and DNA from RNA/DNA deoxyribonucleotide from double-stranded DNA Registered Registered Registered for 1 hour at 37°C in a 50 µl reaction released Quality Environmental Medical Devices hybrids in the 5´ to 3´ direction but is unable to in a total reaction volume of 50 µl in 30 minutes Management Management 15 nmol adenosine-5-monophosphate. degrade either double-stranded or single-stranded at 25°C RNA (2). The protein is the product of T7 gene 6. CERTIFICATE OF ANALYSIS Source: Purified from an E. coli strain containing a Unit Assay Conditions: 50 mM potassium Physical Purity: Purified to > 95% homogeneity TYB12 intein fusion acetate, 20 mM Tris-acetate, 10 mM magnesium as determined by SDS-PAGE analysis using T7 Exonuclease acetate, 1mM dithiothreitol (pH 7.9) and 0.15 mM Coomassie Blue detection. Supplied in: 10 mM Tris-HCl (pH 8.0), sonicated duplex [3H] DNA. 0.1 mM EDTA, 5 mM DTT and 50% glycerol. RNase Activity (Extended Digestion): A 10 µl Quality Control Assays 1-800-632-7799 reaction in NEBuffer 4 containing 40 ng of [email protected] Reagents Supplied with Enzyme: Single Stranded Deoxyribonuclease Activity flourescein labeled RNA transcript and 10 units www.neb.com 10X NEBuffer 4. (FAM Labeled Oligo): A 50 µl reaction in of T7 Exonuclease incubated at 37°C. After M0263S 003121214121 NEBuffer 4 containing a 20 nM solution of a incubation for 4 hours, > 90% of the substrate Reaction Conditions: fluorescent internal labeled oligonucleotide and a RNA remains intact as determined by gel minimum of 50 units of T7 Exonuclease incubated 1X NEBuffer 4. electrophoresis using fluorescence detection. M0263S Incubate at 25°C. for 16 hours at 37°C yields < 5% degradation as determined by capillary electrophoresis. 1,000 units 10,000 U/ml Lot: 0031212 Heat Inactivation: No 1X NEBuffer 4: RECOMBINANT Store at –20°C Exp: 12/14 50 mM potassium acetate Endonuclease Activity: Incubation of a 50 µl References: reaction containing 100 units of T7 Exonuclease Description: T7 Exonuclease acts in the 5´ to 20 mM Tris-acetate 1. Kerr, C. and Sadowski, P.D. (1972) J. Biol. with 1 µg of φX174 RF I DNA for 4 hours at 25°C 3´ direction, catalyzing the removal of 5´ mono- 10 mM magnesium acetate Chem. 247, 305–318. resulted in < 10% loss in supercoiled DNA as nucleotides from duplex DNA. T7 Exonuclease 1 mM DTT 2. Shinozaki, K. and Okazaki, T. (1978) Nucleic determined by agarose gel electrophoresis. is able to initiate nucleotide removal from the pH 7.9 @ 25°C Acids Res. 5, 4245–4261. 5´ termini or at gaps and nicks of double-stranded Functional Assay (RNase, RNA/DNA Hybrid): DNA (1). It will degrade both 5´ phosphorylated Unit Definition: One unit is defined as the amount Incubation of 10 units of T7 Exonuclease with or 5´ dephosphorylated DNA. It has been also of enzyme to produce 1 nmol of acid soluble 20 nmol [3H]poly(A).poly(dT) hybrid polymer ISO 9001 ISO 14001 ISO 13485 reported to degrade RNA and DNA from RNA/DNA deoxyribonucleotide from double-stranded DNA Registered Registered Registered for 1 hour at 37°C in a 50 µl reaction released Quality Environmental Medical Devices hybrids in the 5´ to 3´ direction but is unable to in a total reaction volume of 50 µl in 30 minutes Management Management 15 nmol adenosine-5-monophosphate. degrade either double-stranded or single-stranded at 25°C RNA (2). The protein is the product of T7 gene 6. CERTIFICATE OF ANALYSIS.