New England Biolabs, M0568S, Thermolabile Exonuclease I

Interested in a 5-minute protocol? Try out the Exo-CIP Related Categories Exonucleases and Non-specific Endonucleases Specification Unit Definition One unit of Thermolabile Exonuclease I is defined as the amount of enzyme that will catalyze the release of 2 nmol of acid-soluble nucleotide in a total reaction volume of 100 μl in 6 minutes at 37°C in 50 mM Tris-HCl (pH 7.9), 100 mM NaCl, 10 mM MgCl 2 and 100 μg/ml BSA with 0.17 mg/ml single-stranded [3H]- E. coli DNA. Reaction Conditions 1X NEBuffer™ r3.1 Incubate at 37°C 1X NEBuffer™ r3.1 100 mM NaCl 50 mM Tris-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Storage Buffer 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 0.25 M NaCl 50% Glycerol 200 µg/ml BSA pH 7.4 @ 25°C Heat Inactivation 80°C for 1 minute Unit Assay Conditions 50 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 100 mM NaCl, 100 μg/ml BSA and 0.17 mg/ml single-stranded [3H]- E.coli DNA. FAQ Q: How can I remove the remaining primers and dNTPs from a PCR reaction prior to DNA sequencing? A: In a typical protocol, one can mix 5 µl of PCR reaction (from 30-35 PCR cycles), 1 µl of Thermolabile Exonuclease I and 1 µl Quick CIP (no need to add rCutSmart Buffer) together, followed by incubation at 37°C for 4 minutes, then at 80°C for 1 minute to inactivate the enzyme. Store the cleanup sample at -20°C prior sequencing. Use 3 µl of the cleanup product for Sanger-base sequencing. Q: Can Thermolabile Exonuclease I remove primers in nested PCR reactions? A: Yes, add 1 µl of Thermolabile Exonuclease I to first round of PCR products (up to 20 µl) and incubate the reaction at 37°C for 10 minutes, followed by 80oC 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: Will Thermolabile Exonuclease I degrade RNA? A: No, RNA is not a substrate for Thermolabile Exonuclease I when used as recommended. Q: Can Thermolabile 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 Thermolabile Exonuclease I be heat inactivated? A: Yes. Heat inactivate the enzyme at 80°C for 1 minute, 65°C for 5 minutes or 60°C for 15 minutes. Q: Can Thermolabile 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 Thermolabile Exonuclease I work in other buffers? A: Yes, incubation for 4 minutes at 37°C in the following buffers resulted in removal of > 95% of oligos (20 pmol of 20mer ssDNA): Buffers % of removed oligos Standard Taq Reaction Buffer > 95 % LongAmp® Taq Reaction Buffer > 95 % OneTaq® Standard Reaction Buffer > 95 % Isothermal Amplification Buffer > 95 % Thermopol Reaction Buffer > 95 % Epimark® Hot Start Taq Reaction Buffer > 95 % Q5® Reaction Buffer > 95 % AmpliTaq® 360 (Gold) Buffer > 95 % Phusion® HF Buffer > 95 % Green GoTaq® Reaction Buffer > 95 % NEBuffer® r1.1 > 95 % NEBuffer r2.1 > 95 % NEBuffer r3.1 > 95 % rCutSmartTM Buffer > 95 % Q: Can Exonuclease I be used with a double stranded exonuclease to clean up plasmid preparations? A: Exonuclease I can be used with Lambda Exonuclease (NEB# M0262) to clean up plasmid preps. Exonuclease III (NEB# M0206) and T7 Exonuclease (NEB# M0263) will also work, but will damage nicked plasmids. Although Exonuclease I can be used, we recommend using Exonuclease V (RecBCD) (NEB #M0345) to remove chromosomal DNA after plasmid prep (see our Application Note: Using Exonuclease V (RecBCD) to Eliminate Residual Genomic DNA When Purifying Low Copy Plasmids with the Monarch® Plasmid Miniprep Kit. Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price.