New England Biolabs, M5508S, Thermolabile USER® II Enzyme

A thermostable version is also available, Thermostable USER III Enzyme ( Related Categories DNA Repair Enzymes and Structure-specific Endonucleases Applications Applications of USER® and Thermolabile USER II Enzymes,, USER, ®, Cloning,, DNA Assembly and Cloning, Specification Unit Definition One unit is defined as the amount of enzyme required to nick 10 pmol of a fluorescently labeled 34 mer oligonucleotide duplex containing a single uracil base in 15 minutes at 37°C in a total reaction volume of 10 μl in 1X T4 DNA Ligase Buffer. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 37°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) Activity in NEBuffers Storage Buffer 15 mM Tris-HCl 35 mM NaCl 25 mM KCl 1 mM DTT 0.1 mM EDTA 100 µg/ml BSA 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 65°C for 10 minutes FAQ Q: What is the activity of Thermolabile USER II Enzyme in other buffers? A: Thermolabile USER II Enzyme is 100% active in a variety of buffers, including rCutSmart Buffer (NEB #B6004), NEBuffer r1.1, T4 DNA Ligase Reaction Buffer (NEB #B0202) and TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). Activity in NEBuffers is as follows: NEBuffer r1.1 r2.1 r3.1 rCutSmart™ % Activity 100 100 100 100 Additionally, Thermolabile USER II Enzyme is 100% active in many polymerase and PCR buffers (see chart below). Buffer % Activity Thermopol Reaction Buffer (NEB #B9004) 100% Q5® Reaction Buffer (NEB #B9027) 100% Phusion™ HF Buffer (NEB #B0518) 100% PCR Buffer (Roche) 100% GoTaq® Buffer (Promega®) 100% PCR Buffer (Qiagen®) 100% Pfu Turbo Cx Buffer (Agilent Technologies) 100% AmpliTaq Gold® 360 Buffer (ThermoFisher®) 100% Fast Start™ Taq DNA Pol PCR Buffer (Sigma Aldrich) 100% PCR Buffer (Applied Biosystems) 100% PCR Buffer (Enzymatics) 100% AmpliTaq Gold®, Phusion® and Thermo Fisher® are registered trademarks and property of Thermo Fisher Scientific. s.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: Can Thermolabile USER Enzyme be heat inactivated? A: Yes, unlike USER Enzyme (NEB #M5505), which cannot be heat inactivated and must be removed by other methods (e.g., column purification/phenol chloroform extraction), Thermolabile USER Enzyme can be heat inactivated at 65°C for 10 min. Q: How is Thermolabile USER® II Enzyme different than USER Enzyme? A: Thermolabile USER II Enzyme is similar to USER (Uracil-Specific Excision Reagent) Enzyme (NEB #M5505), in that both will generate a single nucleotide gap at the location of a uracil, with the added benefit of being fully heat inactivated at 65°C. The lyase activity of Endonuclease VIII in USER Enzyme breaks the phosphodiester backbone and leaves a 3´ and 5´ phosphate, whereas the lyase activity of Endonuclease III in Thermolabile USER II Enzyme breaks the phosphodiedester backbone and leaves a 3´-phospho-α, β-unsaturated aldehyde and a 5´ phosphate. Q: Will Thermolabile USER II Enzyme (NEB #M5508) remove uracil from ssDNA and dsDNA? A: Thermolabile USER II Enzyme will remove uracil from both ssDNA and dsDNA, however, Thermolabile USER II Enzyme has ~ 3-fold lower activity on ssDNA as compared to dsDNA. Thermolabile USER II Enzyme has no activity on nucleotides (dUTP) in solution. Q: What applications are Thermolabile USER® II Enzyme best suited for? A: Thermolabile USER II Enzyme can be used in many applications that currently employ USER Enzyme (NEB #M5505), such as USER cloning, cleavage of NEBNext® adaptors during NGS library preparation, and selective removal of the dU-containing second strand during the NEBNext Directional RNA-seq workflow. Please visit Applications of User and Thermolabile USER II Enzymes for more information. Q: During an NEBNext DNA library prep, which enzyme cleaves NEBNext DNA loop adaptors and uracil-containing single stranded loops? A: USER Enzyme is made from UDG and Endonuclease VIII. The UDG helps to create a gap at the site of the uracil and Endonuclease VIII goes to that site and cleaves the phosphodiester backbone at the 3´ and 5´ sides releasing base-free deoxyribose. 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. 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 Thermolabile USER II Enzyme (NEB #M5508) cleave? A: Thermolabile USER® II 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. Thermolabile USER II Enzyme will cleave a deoxyuracil in as little as 3 base pairs from the 5’ end of double-stranded or at least 6 nucleotides from the 3’ end of single-stranded DNA. Please refer to Cleavage of deoxyuracil close to the ends of dsDNA and ssDNA for more information.