New England Biolabs, M0379L, Exonuclease VII

Related Categories Exonucleases and Non-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme that will catalyze the release of 1 nmol of acid-soluble nucleotide in a total reaction volume of 50 μl in 30 minutes at 37°C. Reaction Conditions 1X Exonuclease VII Reaction Buffer Incubate at 37°C 1X Exonuclease VII Reaction Buffer 50 mM Tris-HCl 50 mM Sodium Phosphate 8 mM EDTA 10 mM 2-mercaptoethanol (pH 8 @ 25°C) Storage Buffer 50% Glycerol 50 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 0.1 M NaCl 0.1% Triton® X-100 pH 7.5 @ 25°C Heat Inactivation 95°C for 10 minutes Unit Assay Conditions 1X Exonuclease VII Reaction Buffer containing 0.15 mM sonicated single-stranded [ 3 H] E. coli DNA. FAQ Q: What is the activity of Exonuclease VII in the following buffers? A: Buffers Activity (%) Standard Taq Reaction Buffer 100 Crimson Taq Reaction Buffer 50 LongAmp® Taq Reaction Buffer 100 OneTaq® Stardand Reaction Buffer 100 Isothermal Amplification Buffer 75 Thermopol® Reaction Buffer 75 Epimark® Hot Start Taq Reaction Buffer 100 Q5® Reaction Buffer 75 Q5 Reaction Buffer + enhancer 60 T4 DNA Ligase Buffer 50 Phusion™ HF Buffer 90 Green GoTaq® Reaction Buffer 25 NEBuffer 1 10 NEBuffer 2 75 NEBuffer 3 75 NEBuffer 4 75 Cutsmart™ 75 Q: Will Exonuclease VII work in a PCR buffer? A: Yes. Exonuclease VII works well in most PCR buffers as shown in the following table. Q: What is the molecular weight of the Exonuclease VII? A: The large subunit is 51.8 kDa and small subunit is 8.8 kDa. Q: Is Exonuclease VII a tagged protein? A: No. Exonuclease VII is not a tagged protein. Q: Does Exonuclease VII cut RNA? A: No, Exonuclease VII should not act on an RNA substrate. Q: Can DNA be blunted using Exonuclease VII? A: No, short ends 4 bp or less are not good substrates. Use DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) to fill in 5' overhangs (also called 3' recessed ends) and chew back 3' overhangs or use Mung Bean Nuclease (NEB# M0250) to chew back 3' or 5' overhangs. Note: 3' overhangs cannot be filled in. Q: What is the difference between Exonuclease VII and Exonuclease I (NEB# M0293)? A: Exonuclease VII can degrade ssDNA from both directions unlike Exonuclease I which works only from the 3' to 5' direction. Exonuclease VII can also degrade both 5' or 3' terminally phosphorothioated ssDNA-oligos. Exonuclease I is blocked by the presence of 3’ phosphorothioates. Q: How to remove primers from a PCR reaction? A: In a typical protocol, we mix 5 ul of PCR products and 2 ul of Exonuclease VII together, followed by incubation at 37°C for 30 min and 95°C for 10 min.