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For general laboratory use. FOR IN VITRO USE ONLY. T4 DNA Polymerase From T4 plasmid pTL43W infected Escherichia coli 71–18 Deoxynucleoside-triphosphate: DNA deoxynucleotidyl-transferase (DNA-directed), EC 2.7.7.7 Cat. No. 11 004 786 001 Cat. No. 11 004 794 001 100 U 500 U Product overview Pack content Vial Product description Enzyme properties Content T4 DNA polymerase (1 U/l) • 100 l (100 U pack size) • 500 l (500 U pack size) Enzyme storage buffer; 10 mM TrisHCl, 100 mM NaCl, 2.5 mM MgCl2, 0.5 mM EDTA, 2 mM dithioerythritol, 50% glycerol (v/v), pH 8.0 (4 °C) Incubation buffer, 5 conc. • 1 ml ( 100 U pack size) • 1 ml (500 U pack size) Buffer composition: 250 mM Tris-HCl, 75 mM (NH4)SO4, 35 mM MgCl2, 0.5 mM EDTA, 50 mM 2-mercaptoethanol, 0.1 mg/ml bovine serum albumin (BSA), pH 8.8 (25°C) For polymerization reactions, supplement the buffer with dATP*, dCTP*, dGTP* and dTTP*, 33 mol/l each (final concentration). This buffer is optimal for the polymerase and exonuclease activities of T4 DNA polymerase T4 DNA polymerase is a DNA dependent DNA polymerase that catalyzes the polymerization of deoxynucleoside-5´-triphosphates to the hydroxyl termini of recessive ends. Blunt ended DNA cannot serve as template for the polymerization reaction. For polymerization T4 DNA polymerase requires DNA with 5´protruding ends and a high concentration of dNTP’s. The enzyme carries an extremely active 3´→5´-exonuclease that shows a strong specificity for ssDNA (1) and lacks a 5´→3´-exonuclease activity. Therefore nicked duplex DNA cannot serve simultaneously as template and primer for polymerization. The addition of T4 gene 32 protein facilitates strand displacement and therefore allows T4 DNA polymerase to replicate the nicked duplex (2,3). Note: Low levels of dNTP’s should not be used for polymerization reaction because once the dNTP’s are exhausted, the exonuclease activity will degrade the DNA. Volume activity ≥ 1 U/l of T4 DNA polymerase is the amount of enzyme activity that incorporates 10 nmol dNTP into acid-precipitable DNA products in 30 min at 37°C( 4). For lot-specific values see data label. Specific activity ≥ 5 U/g according to Gouliand and Bradford (4,5). For lot-specific values see data label Purity ≥ 10 g of T4 DNA polymerase migrate as a single band in SDSpolyacrylamide gel electrophoresis according to Laemmli (6). Mass weight single polypeptide of 114 000 Optimal pH 8 9 (at pH 7.5 and 9.7 approx. 50% activity is found) Optimal Mg2+ approx. 6 mM 0904.11006835 5 Version Sept. 2004 Store at 15 to 25° C Storage and stability The undiluted enzyme solution is stable at -15 to -25°C until the control date printed on the label. Application T4 DNA polymerase is used to label 3´-termini of DNA. Extensive labeling is achieved by the replacement reaction, in which the 3´-exonuclease activity of the enzyme first digests dsDNA to produce molecules with recessed 3´-termini (7). On subsequent addition of labeled dNTPs, the polymerase activity of T4 DNA polymerase then extends the 3´-ends along the length of the template. Exonuclease III from E. coli can be used to create partially single-stranded dsDNA for subsequent polymerization reactions (8). Molecules labeled to high specific activity are used chiefly as hybridization probes. They have two advantages over probes prepared by nick translation: they lack artificial hairpin structures and they can easily be converted into strand-specific probes by cleavage with suitable restriction endonucleases (9). In combination with T4 gene 32 protein T4 DNA polymerase is used for gap-filling in site-directed mutagenesis experiments (10). Quality control See data label for lot-specific values. Test buffer 50 mM Tris-HCl, 7 mM MgCl2, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, 15 mM (NH4)2SO4, 0.1 mg/ml BSA, pH approx. 7.5 (at 37° C). Absence of endonucleases 1. 1 g DNA is incubated with T4 DNA polymerase for 4 h at 37° C in 50 l test buffer. The number of enzyme units which show no degradation of DNA is stated under “Endo 1”. 2. 1 g Eco RI/Hind III fragments of DNA is incubated with T4 DNA polymerase for 4 h at 37° C in 50 l test buffer. The number of enzyme units which show no alteration of the banding pattern is stated under “Endo 2”. Absence of ssspecific DNases 1 g ss M13mp9 DNA are incubated with T4 DNA polymerase for 4 h at 37° C in 50 l test buffer. The number of enzyme units that show no degradation of ss M13mp9 DNA is stated under “ss-Act”. Absence of nicking activity 1 g pBR322 DNA is incubated with T4 DNA polymerase for 4 h at 37° C in 50 l test buffer. The number of enzyme units which show no relaxing of supercoiled structure is stated unter “Nick. Act.” Absence of RNases 4 g MS2 RNA are incubated with T4 DNA polymerase for 4 h at 37° C in 50 l test buffer. The number of enzyme units which produce no change in the appearance of MS2 RNA is stated under “RNase”. References 1 2 3 4 5 6 7 Huang, W. M. & Lekman, I. R. (1972) J. Biol. Chem. 247, 3139. Alberts, B. & Frey, L. (1970) Nature 227, 1313. Nossal, N. G. (1974) J. Biol. Chem. 249, 5668. Goulian, M. et al. (1968) J. Biol. Chem. 243, 627. Bradford, M. (1976) Anal. Biochem. 72, 248. Laemmli, U. K. (1970) Nature 227, 680. Maniatis, T. et al. (1989) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, New York. 8 Richardson, C. C. & Kornberg, A. (1964) J. Biol. Chem. 244, 2996. 9 Deen, K. D. et al. (1983) Anal. Biochem. 135, 456. 10 Craik, C. S. et al. (1985) Science 228, 291. Ordering Information Roche Applied Science offers a large selection of reagents and systems for life science research. For a complete overview of related products and manuals, please visit and bookmark our homepage at: http://www.roche-applied-science.com and our Special Interest Sites including: • DIG Reagents and Kits for Non-Radioactive Nucleic Acid Labeling and Detection: http://www.rocheapplied-science.com/DIG/ Product Pack size Cat. No. 100 µg 500 µg 10 972 983 001 10 972 991 001 DIG-Nick Translation Mix 60 µl 11 745 816 001 Biotin-Nick Translation Mix 160 µl 11 745 824 001 Nick Translation Mix 200 µl 11 745 808 001 T4 Gene 32 Protein Nick Translation Kit 1 kit 10 976 776 001 (50 radioactive reactions) DNA Polymerase I 500 units 1000 units 10 104 485 001 10 104 493 001 DNA Polymerase I, endonuclease-free 250 units 1000 units 10 642 711 001 10 642 72 0001 DNase I, RNase-free 10000 units 10 776 785 001 How to contact Roche Applied Science www.roche-applied-science.com to order, solve technical queries, find product information, or contact your local sales representative. www.roche-applied-science.com/pack-insert/11004786001a.pdf Please visit our new Online Technical Support Site under www.roche-applied-science.com/support Roche Diagnostics GmbH Roche Applied Science Nonnenwald 2 82372 Penzberg Germany