New England Biolabs, M0527S, Msz Exonuclease I

Related Categories Exonucleases and Non-specific Endonucleases Specification Unit Definition One unit of Msz Exonuclease I is defined as the amount of enzyme that will catalyze the release of 5 nmol of acid-soluble nucleotide in a total reaction volume of 100 μl in 15 minutes at 55°C in 1X rCutSmart Buffer. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 55°C 1X rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Heat Inactivation 80°C for 1 minute FAQ Q: What is the optimal reaction temperature range for Msz Exonuclease I activity? A: Msz Exonuclease I is most active between 50°C - 58°C. Temperatures below 45°C or above 60°C are not recommended. Q: Can Msz Exonuclease I remove primers in nested PCR reactions? A: Yes, add 1 µl of Msz Exonuclease I to the first round of PCR products (up to 20 µl) and incubate the reaction at 55°C for 15 minutes, followed by 80°C for 1 min to inactivate the enzyme. New sets of primers can then be added followed by a second round of PCR to reduce the nonspecific PCR amplicons. Q: Can Msz Exonuclease I be used to blunt dsDNA? A: No. Short ssDNA ends are not a substrate. Use DNA Polymerase I, Large Klenow Fragment (NEB# M0210) to fill in 5′ overhangs 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: Can Msz Exonuclease I be heat inactivated? A: Yes. Heat inactivate the enzyme at 80°C for 1 minute. Q: Can Msz Exonuclease I be used with a double-stranded exonuclease to clean up plasmid preparations? A: This is not recommended. The best enzyme to cleanup genomic DNA contamination in plasmid preparations is Exonuclease V (RecBCD, NEB #M0345). Q: Will Msz Exonuclease I work in other buffers? A: Yes. Most PCR buffers are compatible. No additional reaction buffer is necessary if the enzyme is added to a PCR reaction. Incubation in the following buffers at 55°C for 15 minutes resulted in the relative activity listed: NEBuffer % Activity r1.1 (NEB# B7030) 20 r2.1 (NEB# B6002) 50 r3.1 (NEB# B6003) 50 rCutSmartTM (NEB# B6004) 100 Exonuclease I Reaction Buffer (NEB# B0293) 10 ThermoPol (NEB# B9004) 90