Source: An E. coli strain containing plasmids Unit Assay Conditions: 1X NEBuffer 4, 1 mM ATP Physical Purity: Purified to > 95% homogene- Exonuclease V for expressing the three subunits of E. coli with 0.15 mM sonicated duplex [3H]-DNA. ity as determined by SDS-PAGE analysis using Exonuclease V: RecB, RecC and RecD. Coomassie Blue detection. (RecBCD) Heat Inactivation: 70°C for 30 minutes. Supplied in: 100 mM NaCl, 50 mM Tris-HCl A Typical Exonuclease V Reaction: 1-800-632-7799 (pH 7.5 @ 25°C), 0.1 mM EDTA, 1 mM DTT, Quality Control Assays x µl sample DNA (~ 1 µg) [email protected] 0.1% Triton X-100 and 50% glycerol. 3 µl NEBuffer4 (10X) www.neb.com Endonuclease Activity I: Incubation of a 50 µl 3 µl 10 mM ATP M0345S 001121014101 reaction containing 100 units of Exonuclease V Reagents Supplied with Enzyme: y µl H20 (up to final volume of 30 µl) 10X NEBuffer 4, 10 mM ATP with 1 µg of φX174 RF I DNA in NEBuffer 4 and 1 µl Exonuclease V (10 units) 1 mM ATP for 4 hours at 37°C resulted in < 10% M0345S Reaction Conditions: 1X NEBuffer 4 loss in φX174 RF I DNA as determined by agarose 1. Incubate at 37°C for 30 minutes. 2. To stop reaction add EDTA to 11 mM. 1,000 units 10,000 U/ml Lot: 0011210 supplemented with 1 mM ATP. Incubate at 37°C. gel electrophoresis. 3 Heat Inactivation 70°C for 30 minutes. RECOMBINANT Store at –20°C Exp: 10/14 1X NEBuffer 4: Endonuclease Activity II: Incubation of a 50 µl 4. Clean-up treated samples by column purifica- Description: Exonuclease V, a RecBCD complex 50 mM potassium acetate reaction containing 50 units of Exonuclease V with tion and/or ethanol precipitation. 20 mM Tris-acetate 1 µg of φX174 RF II DNA in NEBuffer 4 and 1 mM from E. coli has several different enzyme activities, Note: Estimate amount of DNA to be removed by 10 mM magnesium acetate ATP for 4 hours at 37°C resulted in < 10% loss in including an ATP-dependent single-stranded DNA agarose gel electrophoresis or OD . If > 1 µg 1 mM dithiothreitol φX174 RF II DNA as determined by agarose gel 260 endonuclease activity, ss- and ds- DNA exo- scale up all reaction components proportionately. nuclease activity. The hydrolysis in each case is pH 7.9 @ 25°C electrophoresis. bi-directional (from both the 3´ and 5´ ends) and processive, producing oligonucleotides (1,2,3). Unit Definition: One unit is defined as the amount RNase Assay: Incubation of 10 units of enzyme (see other side) All Exonuclease V activities have divalent cation of enzyme required to produce 1 nmol of acid- with 40 ng of 300 bases Fam-labeled RNA at requirements. Mg2+ is required for the exonuclease soluble deoxyribonucleotide from double-stranded 37°C for 3 hours in a 50 µl reaction buffer results activity, while Ca2+ inhibits the exonuclease activ- DNA in 30 minutes at 37°C in a total reaction in < 10% of RNA degradation as analyzed from ity and allows double-stranded DNA unwinding volume of 50 µl. polyacrylamide gel. (helicase activity) without hydrolysis (4,5). CERTIFICATE OF ANALYSIS Source: An E. coli strain containing plasmids Unit Assay Conditions: 1X NEBuffer 4, 1 mM ATP Physical Purity: Purified to > 95% homogene- for expressing the three subunits of E. coli with 0.15 mM sonicated duplex [3H]-DNA. ity as determined by SDS-PAGE analysis using Exonuclease V Exonuclease V: RecB, RecC and RecD. Coomassie Blue detection. (RecBCD) Heat Inactivation: 70°C for 30 minutes. Supplied in: 100 mM NaCl, 50 mM Tris-HCl A Typical Exonuclease V Reaction: 1-800-632-7799 (pH 7.5 @ 25°C), 0.1 mM EDTA, 1 mM DTT, Quality Control Assays x µl sample DNA (~ 1 µg) [email protected] 0.1% Triton X-100 and 50% glycerol. 3 µl NEBuffer4 (10X) www.neb.com Endonuclease Activity I: Incubation of a 50 µl 3 µl 10 mM ATP M0345S 001121014101 reaction containing 100 units of Exonuclease V Reagents Supplied with Enzyme: y µl H20 (up to final volume of 30 µl) 10X NEBuffer 4, 10 mM ATP with 1 µg of φX174 RF I DNA in NEBuffer 4 and 1 µl Exonuclease V (10 units) 1 mM ATP for 4 hours at 37°C resulted in < 10% 1. Incubate at 37°C for 30 minutes. M0345S Reaction Conditions: 1X NEBuffer 4 loss in φX174 RF I DNA as determined by agarose 2. To stop reaction add EDTA to 11 mM. 1,000 units 10,000 U/ml Lot: 0011210 supplemented with 1 mM ATP. Incubate at 37°C. gel electrophoresis. 3 Heat Inactivation 70°C for 30 minutes. RECOMBINANT Store at –20°C Exp: 10/14 1X NEBuffer 4: Endonuclease Activity II: Incubation of a 50 µl 4. Clean-up treated samples by column purifica- tion and/or ethanol precipitation. Description: Exonuclease V, a RecBCD complex 50 mM potassium acetate reaction containing 50 units of Exonuclease V with 20 mM Tris-acetate 1 µg of φX174 RF II DNA in NEBuffer 4 and 1 mM from E. coli has several different enzyme activities, Note: Estimate amount of DNA to be removed by 10 mM magnesium acetate ATP for 4 hours at 37°C resulted in < 10% loss in including an ATP-dependent single-stranded DNA agarose gel electrophoresis or OD . If > 1 µg 1 mM dithiothreitol X174 RF II DNA as determined by agarose gel 260 endonuclease activity, ss- and ds- DNA exo- φ scale up all reaction components proportionately. nuclease activity. The hydrolysis in each case is pH 7.9 @ 25°C electrophoresis. bi-directional (from both the 3´ and 5´ ends) and Unit Definition: One unit is defined as the amount RNase Assay: Incubation of 10 units of enzyme processive, producing oligonucleotides (1,2,3). (see other side) All Exonuclease V activities have divalent cation of enzyme required to produce 1 nmol of acid- with 40 ng of 300 bases Fam-labeled RNA at requirements. Mg2+ is required for the exonuclease soluble deoxyribonucleotide from double-stranded 37°C for 3 hours in a 50 µl reaction buffer results activity, while Ca2+ inhibits the exonuclease activ- DNA in 30 minutes at 37°C in a total reaction in < 10% of RNA degradation as analyzed from ity and allows double-stranded DNA unwinding volume of 50 µl. polyacrylamide gel. (helicase activity) without hydrolysis (4,5). CERTIFICATE OF ANALYSIS References: 1. Eichler, D.C. et al. (1977) J. Biol. Chem. 252, 499–503. 2. Tayler, A.F. et al. (1985) J. Mol. Biol.185, 431–443. 3. Amundsen, S.K. et al. (1986) Proc. Natl. Acad. Sci. 83, 5558–5562. 4. Palas, K.M. et al. (1990) J. Biol. Chem. 265, 3447–3454. 5. Dillingham, M.S. et al. (2008) Microbiology and Mol. Biol. Review. 72, 642–671. Page 2 (M0345) References: 1. Eichler, D.C. et al. (1977) J. Biol. Chem. 252, 499–503. 2. Tayler, A.F. et al. (1985) J. Mol. Biol.185, 431–443. 3. Amundsen, S.K. et al. (1986) Proc. Natl. Acad. Sci. 83, 5558–5562. 4. Palas, K.M. et al. (1990) J. Biol. Chem. 265, 3447–3454. 5. Dillingham, M.S. et al. (2008) Microbiology and Mol. Biol. Review. 72, 642–671. Page 2 (M0345).