New England Biolabs, M5509S, Thermostable USER® III Enzyme

Thermostable USER (Uracil-Specific Excision Reagent) III Enzyme generates a single nucleotide gap at the location of a uracil residue. It is active between 50-75°C (optimal activity observed at 65°C). Related Categories DNA Repair Enzymes and Structure-specific Endonucleases Applications DNA Assembly and Cloning,, High-throughput cloning and automation solutions Specification Unit Definition One unit is defined as the amount of enzyme required to nick 10 pmol of a fluorescently labeled 60 mer oligonucleotide duplex containing a single uracil base, in 15 minutes at 65°C in a total reaction volume of 10 μl in 1X Thermopol Buffer. Reaction Conditions 1X ThermoPol® Reaction Buffer Incubate at 65°C 1X ThermoPol® Reaction Buffer 20 mM Tris-HCl 10 mM (NH4)2SO4 10 mM KCl 2 mM MgSO4 0.1% Triton® X-100 (pH 8.8 @ 25°C) Storage Buffer 15 mM Tris-HCl 35 mM NaCl 1 mM DTT 0.1 mM 100 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C FAQ Q: What is the activity of Thermostable USER® III Enzyme in other buffers? A: Thermostable USER III Enzyme is 100% active in Thermopol Buffer and has limited activity in other buffers. Activity in NEBuffers at 65°C is as follows: NEBuffer r1.1 r2.1 r3.1 rCutSmart™ % Activity 100 50 50 75 Additionally, Thermolabile USER II Enzyme is 100% active in many polymerase and PCR buffers (see chart below). Buffer Activity (Relative to Thermopol Buffer) Thermopol II Reaction Buffer (NEB #B9004) 100% Q5® Reaction Buffer (NEB #B9027) 50% Colorless GoTaq® Flexi Buffer (Promega®) 75% PCR Buffer (Qiagen®) 75% iTaq Buffer 100% AmpliTaq Gold® 360 Buffer (ThermoFisher®) 100% Fast Start™ Taq DNA Pol PCR Buffer (Sigma Aldrich) 0% Ex Taq Buffer (Takara) 0% Dream Taq Buffer (ThermoFisher®) 75% Platinum II PCR Buffer (Invitrogen) 75% AmpliTaq Gold® and Thermo Fisher® are registered trademarks and property of Thermo Fisher Scientific. GoTaq® and Promega® are registered trademarks of Promega, Inc. Qiagen® is a registered trademark of Qiagen, Inc. Fast Start™ is a trademark of Sigma Aldrich. Q: What temperature is Thermostable USER® III Enzyme active? A: Thermostable USER III Enzyme is active at temperatures >50°C, with optimal activity observed at 65°C. Minimal activity is observed at temperatures less than 50°C. Like USER Enzyme (NEB #M5505), Thermostable USER III Enzyme cannot be heat inactivated and must be removed by other methods (e.g., column purification/phenol chloroform extraction). Q: How is Thermostable USER® III Enzyme different than USER Enzyme and Thermolabile USER II Enzyme? A: Thermostable USER III Enzyme is similar to USER (Uracil-Specific Excision Reagent) Enzyme (NEB #M5505) and Thermolabile USER II Enzyme (NEB #M5508), in that all 3 will generate a single nucleotide gap at the location of a uracil. Thermolabile USER II Enzyme has the added benefit of being heat-inactivatable (65°C for 10 minutes), while Thermostable USER III Enzyme is active at higher temperatures (>50°C). Thermolabile USER II can be heat inactivated, while USER Enzyme and Thermostable USER III Enzyme must be removed from the reaction via other means (column cleanup or other purification methods). All USER enzymes will break the phosphodiester backbone but leave different 3′ and 5′ ends: the lyase activity of Endonuclease VIII in USER Enzyme leaves a 3′ phosphate and 5′ phosphate, the lyase activity of Endonuclease III in Thermolabile USER II Enzyme leaves a 3′-phospho-α, β-unsaturated aldehyde and 5′ phosphate and the lyase activity of Endonuclease IV in Thermostable USER III Enzyme leaves a 3′-hydroxyl and 5′-deoxyribose phosphate. USER Enzyme (NEB #M5505), Thermolabile USER II Enzyme (NEB #M5508) and Thermostable USER III Enzyme (NEB #M5509) generate different functional ends after cleavage of DNA. In addition to different optimal reaction temperatures (37°C for USER and Thermolabile USER II Enzyme and 65°C for Thermostable USER III Enzyme) and ability to be heat inactivated (Thermolabile USER II Enzyme only), the different USER Enzymes generate different 3’ and 5’ termini after cleavage. USER Enzyme (NEB #M5505) contains Endonuclease VIII and leaves a 3’ and 5’ phosphate after cleavage. Thermolabile USER II Enzyme (NEB #M5508) contains Endonuclease III and leaves a 3′-phospho-α, β-unsaturated aldehyde and 5′ phosphate after cleavage. Thermostable USER III Enzyme (NEB #M5509) contains Endonuclease IV and leaves a 3′-hydroxyl and 5′-deoxyribose phosphate. Q: Will Thermostable USER® III Enzyme remove uracil from ssDNA and dsDNA? A: Thermostable USER III Enzyme will remove uracil from ssDNA with the same efficiency as from dsDNA. Thermostable USER III Enzyme has no activity on nucleotides (dUTP) in solution. Q: What is the difference between the suite of USER® enzymes (#M5505, #M5508, #M5509) and Thermostable Endonuclease Q (#M0701)? A: The USER products (USER Enzyme (#M5505), Thermolabile USER II Enzyme (#M5508) and Thermostable USER III Enzyme (#M5509) are a mixture of two enzymes: UDG and an endonuclease. In contrast, Thermostable Endonuclease Q (#M0701) is a single enzyme. Although the USER enzymes and Thermostable Endonuclease Q both recognize and cleave at deoxyuracil in DNA substrates, the resulting products are different. The end products of the USER enzymes are one-nucleotide gaps with varying functional ends based on the type of USER implemented; the deoxyuracil is removed. The products of Thermostable Endonuclease Q are nicks with a 3′-OH and a 5′-phosphate; the deoxyuracil is still present on the 3′ segment of DNA. Q: Do any of the USER enzymes leave ligatable ends when cleaving at a Uracil? A: No, neither USER Enzyme (NEB #M5505), Thermolabile USER II Enzyme (NEB #M5508) nor Thermostable USER III Enzyme (NEB #M5509) will leave ligatable ends when cleaving at a uracil. However, Thermostable Endonuclease Q (NEB #M0701) will nick 5´ to a uracil (leaving the nucleobase intact), but leave ligatable 3´ OH and 5´ P ends after cleavage. Q: How close to the ends of DNA can Thermostable USER III Enzyme (NEB #M5509) cleave? A: Thermostable USER® III Enzyme will cleave a deoxyuracil in as little as 3 base pairs from the 5’ end of double-stranded or at least 12 nucleotides from the 5’ end of single-stranded DNA. Thermostable USER III Enzyme will cleave a deoxyuracil 3 base pairs or nucleotides from the 3’ end of either double-stranded or single-stranded DNA. Please refer to Cleavage of deoxyuracil close to the ends of dsDNA and ssDNA for more information.