DNA Polymerase I (E. coli) DNA Polymerase I (E. coli) Cat. No. 18010-025 Size: 1,000 units Cat. No. 18010-025 Size: 1,000 units Conc.: 10 U/µl Store at -20°C (not frost-free). Conc.: 10 U/µl Store at -20°C (not frost-free). Description: Description: DNA Polymerase I (E. coli) exhibits three activities: a 5' → 3' DNA polymerase, a DNA Polymerase I (E. coli) exhibits three activities: a 5' → 3' DNA polymerase, a 3' → 5' exonuclease, and a 5' → 3' exonuclease. It may be used to fill in a 3' 3' → 5' exonuclease, and a 5' → 3' exonuclease. It may be used to fill in a 3' recessed end of DNA (1) and in nick translation reactions (2). The enzyme is recessed end of DNA (1) and in nick translation reactions (2). The enzyme is purified from E. coli lambda lysogen NM984 (3). purified from E. coli lambda lysogen NM984 (3). Unit Definition: Unit Definition: One unit incorporates 10 nmol of deoxyribonucleotide into acid-precipitable One unit incorporates 10 nmol of deoxyribonucleotide into acid-precipitable material in 30 min at 37°C. material in 30 min at 37°C. Storage Buffer: Storage Buffer: 50 mM potassium phosphate (pH 7.0) 50 mM potassium phosphate (pH 7.0) 100 mM KCl 100 mM KCl 1 mM DTT 1 mM DTT 50% (v/v) glycerol 50% (v/v) glycerol Quality Control: Quality Control: This product has passed the following quality control assays: This product has passed the following quality control assays: absence of detectable endodeoxyribonuclease activity; pancreatic DNase I dependence for nick translation; performance in a nick translation reaction. Doc. Rev.: 092801 Doc. Rev.: 092801 For research use only. Not for use in diagnostic procedures. DNA Polymerase I (E. coli) DNA Polymerase I (E. coli) Cat. No. 18010-025 Size: 1,000 units Cat. No. 18010-025 Size: 1,000 units Conc.: 10 U/µl Store at -20°C (not frost-free). Conc.: 10 U/µl Store at -20°C (not frost-free). Description: Description: DNA Polymerase I (E. coli) exhibits three activities: a 5' → 3' DNA polymerase, a DNA Polymerase I (E. coli) exhibits three activities: a 5' → 3' DNA polymerase, a 3' → 5' exonuclease, and a 5' → 3' exonuclease. It may be used to fill in a 3' 3' → 5' exonuclease, and a 5' → 3' exonuclease. It may be used to fill in a 3' recessed end of DNA (1) and in nick translation reactions (2). The enzyme is recessed end of DNA (1) and in nick translation reactions (2). The enzyme is purified from E. coli lambda lysogen NM984 (3). purified from E. coli lambda lysogen NM984 (3). Unit Definition: Unit Definition: One unit incorporates 10 nmol of deoxyribonucleotide into acid-precipitable One unit incorporates 10 nmol of deoxyribonucleotide into acid-precipitable material in 30 min at 37°C. material in 30 min at 37°C. Storage Buffer: Storage Buffer: 50 mM potassium phosphate (pH 7.0) 50 mM potassium phosphate (pH 7.0) 100 mM KCl 100 mM KCl 1 mM DTT 1 mM DTT 50% (v/v) glycerol 50% (v/v) glycerol Quality Control: Quality Control: This product has passed the following quality control assays: This product has passed the following quality control assays: absence of detectable endodeoxyribonuclease activity; pancreatic DNase I absence of detectable endodeoxyribonuclease activity; pancreatic DNase I dependence for nick translation; performance in a nick translation reaction. dependence for nick translation; performance in a nick translation reaction. Doc. Rev.: 092801 Doc. Rev.: 092801 Page 2 of 2 Page 2 of 2 Functional Assay Conditions: Functional Assay Conditions: Nick translation reaction (4): Nick translation reaction (4): 50 mM Tris-HCl (pH 7.8) 50 mM Tris-HCl (pH 7.8) 5 mM MgCl2 5 mM MgCl2 10 mM 2-mercaptoethanol 10 mM 2-mercaptoethanol 10 µg/ml BSA 10 µg/ml BSA 1 µg DNA 1 µg DNA 200 pg pancreatic DNase I 200 pg pancreatic DNase I 14.1 µCi [3H] dTTP (45 Ci/mmole) 14.1 µCi [3H] dTTP (45 Ci/mmole) 20 µM dATP, dCTP, dGTP 20 µM dATP, dCTP, dGTP 2 units DNA Polymerase I 2 units DNA Polymerase I Reaction Volume: 100 µl Reaction Volume: 100 µl Incubation: 30 to 60 min at 15°C Incubation: 30 to 60 min at 15°C References: References: 1. (1984) FOCUS , 6:1, 6. 1. (1984) FOCUS , 6:1, 6. 2. Kelly, W. S. and Stump, K. H. (1979) J. Biol. Chem. 254, 3206. 2. Kelly, W. S. and Stump, K. H. (1979) J. Biol. Chem. 254, 3206. 3. Maniatis, T., Jeffrey, A. and Kleid, D. G. (1975) Proc. Natl. Acad. Sci. U.S.A. 3. Maniatis, T., Jeffrey, A. and Kleid, D. G. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1184. 72, 1184. 4. Hartman, C. P. and Rabussay, D. (1981) in Gene Amplification and Analysis 4. Hartman, C. P. and Rabussay, D. (1981) in Gene Amplification and Analysis (Chirikjian, J. G., and Papas, T. S., eds.) Vol. 2, p.17, Elsevier/North Holland, (Chirikjian, J. G., and Papas, T. S., eds.) Vol. 2, p.17, Elsevier/North Holland, New York. New York. REFER TO THE GIBCO BRL CATALOGUE AND REFERENCE GUIDE FOR REFER TO THE GIBCO BRL CATALOGUE AND REFERENCE GUIDE FOR NOTES ON CONDITIONS WHICH AFFECT ENZYME ACTIVITY. NOTES ON CONDITIONS WHICH AFFECT ENZYME ACTIVITY. Cat. No. 18010-025 Cat. No. 18010-025 Page 2 of 2 Page 2 of 2 Functional Assay Conditions: Functional Assay Conditions: Nick translation reaction (4): Nick translation reaction (4): 50 mM Tris-HCl (pH 7.8) 50 mM Tris-HCl (pH 7.8) 5 mM MgCl2 5 mM MgCl2 10 mM 2-mercaptoethanol 10 mM 2-mercaptoethanol 10 µg/ml BSA 10 µg/ml BSA 1 µg DNA 1 µg DNA 200 pg pancreatic DNase I 200 pg pancreatic DNase I 14.1 µCi [3H] dTTP (45 Ci/mmole) 14.1 µCi [3H] dTTP (45 Ci/mmole) 20 µM dATP, dCTP, dGTP 20 µM dATP, dCTP, dGTP 2 units DNA Polymerase I 2 units DNA Polymerase I Reaction Volume: 100 µl Reaction Volume: 100 µl Incubation: 30 to 60 min at 15°C Incubation: 30 to 60 min at 15°C References: References: 1. (1984) FOCUS , 6:1, 6. 1. (1984) FOCUS , 6:1, 6. 2. Kelly, W. S. and Stump, K. H. (1979) J. Biol. Chem. 254, 3206. 2. Kelly, W. S. and Stump, K. H. (1979) J. Biol. Chem. 254, 3206. 3. Maniatis, T., Jeffrey, A. and Kleid, D. G. (1975) Proc. Natl. Acad. Sci. U.S.A. 3. Maniatis, T., Jeffrey, A. and Kleid, D. G. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1184. 72, 1184. 4. Hartman, C. P. and Rabussay, D. (1981) in Gene Amplification and Analysis 4. Hartman, C. P. and Rabussay, D. (1981) in Gene Amplification and Analysis (Chirikjian, J. G., and Papas, T. S., eds.) Vol. 2, p.17, Elsevier/North Holland, (Chirikjian, J. G., and Papas, T. S., eds.) Vol. 2, p.17, Elsevier/North Holland, New York. New York. REFER TO THE GIBCO BRL CATALOGUE AND REFERENCE GUIDE FOR REFER TO THE GIBCO BRL CATALOGUE AND REFERENCE GUIDE FOR NOTES ON CONDITIONS WHICH AFFECT ENZYME ACTIVITY. NOTES ON CONDITIONS WHICH AFFECT ENZYME ACTIVITY. Cat. No. 18010-025 Cat. No. 18010-025 .