A thermostable version is also available, Thermostable USER III Enzyme (NEB #M5509).
We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at www.neb.com/BSA-free.
Thermolabile USER (Uracil-Specific Excision Reagent) II Enzyme generates a single nucleotide gap at the location of a uracil residue. It can be 100% inactivated at temperatures >65°C
Antarctic Thermolabile Uracil DNA glycosylase (UDG) catalyzes the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of Endonuclease III breaks the phosphodiester backbone at the 3´and 5´ sides of the abasic site. In addition to generating a different a 3´-terminus than USER Enzyme (a 3´-phospho-α, β-unsaturated aldehyde versus the 3´phosphate left by USER® Enzyme, NEB #M5505), Thermolabile USER II Enzyme (NEB #M5508) can also be completely heat inactivated after 10 minutes at 65°C.
Thermolabile USER® (Uracil-Specific Excision Reagent) II Enzyme generates a single nucleotide gap at the location of a uracil. Thermolabile USER II Enzyme is a mixture of Antarctic Thermolabile Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease III.
Figure 2: 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.
Figure 3. DNA Libraries Prepared with USER Enzyme and Thermolabile USER II Enzyme for Adaptor Cleavage are Equivalent (A) Illumina® libraries were prepared using various amounts of input DNA (1 µg, 100 ng, 5 ng or 0.5 ng) and the NEBNext® DNA Ultra™ II Library Preparation Kit with USER Enzyme or Thermolabile USER II Enzyme for adaptor cleavage. Libraries were PCR amplified using 3, 5, 9 or 12 cycles, respectively, and quantified on an Agilent Bioanalyzer. Thermolabile USER II Enzyme performs similarly to USER Enzyme and has no effect on library yields. (B) DNA sequencing libraries prepared with either USER Enzyme or Thermolabile USER II Enzyme have comparable insert sizes. (C) Comparable GC bias is observed between libraries prepared using either USER Enzyme or Thermolabile USER II Enzyme.
ILLUMINA® is a registered trademark of Illumina, Inc.
AGILENT® and BIOANALYZER® are registered trademarks of Agilent Technologies
This product is related to the following categories:
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)
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Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
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New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
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