New England Biolabs, M5505L, USER® Enzyme

USER Enzyme has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10233215. 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 34 mer oligonucleotide duplex containing a single uracil base, in 15 minutes at 37°C in a total reaction volume of 10 μl. 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) Storage Buffer 10 mM Tris-HCl 5 mM NaCl 1 mM DTT 0.1 mM EDTA 175 µg/ml Recombinant Albumin 50% Glycerol 50 mM KCl pH 7.4 @ 25°C Heat Inactivation No FAQ Q: Will the USER Enzyme work in rCutSmart buffer? A: The USER enzyme 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: Where can I find information or a protocol for cloning with USER® Enzyme? A: https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning 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 USER Enzyme (NEB #M5505) cleave? A: USER® Enzyme will cleave a deoxyuracil in as little as 3 base pairs from the 5’ end of double-stranded or at least 9 nucleotides from the 5’ end of single-stranded DNA. USER Enzyme will cleave a deoxyuracil 6 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.