New England Biolabs, M0263S, T7 Exonuclease

Related Categories Exonucleases and Non-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme required to produce 1 nmol of acid-soluble deoxyribonucleotide in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X NEBuffer 4 with 0.15 mM sonicated duplex [ 3 H]-DNA. Reaction Conditions 1X NEBuffer™ 4 Incubate at 25°C 1X NEBuffer™ 4 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 1 mM DTT (pH 7.9 @ 25°C) Activity in NEBuffers Storage Buffer 10 mM Tris-HCl 5 mM DTT 0.1 mM EDTA 50% Glycerol pH 8 @ 25°C Heat Inactivation No FAQ Q: How Does T7 Exonuclease differ from Lambda Exonuclease (NEB# M0262)? A: T7 Exonuclease has even less activity on single stranded DNA than Lambda Exonuclease, acts at nicks unlike Lambda Exonuclease, and is less processive. Since it is less processive it may be more useful for unidirectional nested deletions. The enzyme can be diluted to control the reaction rate. With a highly processive enzyme, like Lambda Exonuclease, dilution gives a mix of completely digested products and undigested substrate. Q: How Does T7 Exonuclease differ from Exonuclease III (NEB# M0206)? A: Exonuclease III acts in the 3' to 5' direction, opposite that of T7 Exonuclease and Lambda Exonuclease. Q: What is the activity of T7 Exonuclease in the NEBuffers? A: NEBuffer 1: 30% NEBuffer 2: 25% NEBuffer 3: 1% NEBuffer 4: 100% Q: Can T7 Exonuclease be heat inactivated? A: No. Q: What is the best way to generate partials using T7 Exonuclease? A: The enzyme should be titered to the substrate. Serial dilution of the DNA incubated for a specific time is suggested. Start with a 30 minute incubation. Incubating at room temperature can be used to slow the reaction. If the DNA is limiting the enzyme can be diluted in reaction buffer for immediate use. Q: Can DNA be blunted using T7 Exonuclease? A: No. 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 can not be filled in. Q: Will T7 Exonuclease work in rCutSmart buffer? A: Yes, T7 Exonuclease will work in rCutSmart buffer. To see its % functional activity in rCutSmart, and that of other DNA Modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart. 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.