USER Enzyme has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10233215.
Thermolabile USER II Enzyme (NEB #M5508) and Thermostable USER III Enzyme (NEB #M5509) are available.
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.
USER (Uracil-Specific Excision Reagent) Enzyme (1) generates a single nucleotide gap at the location of a uracil residue.
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 VIII breaks the phosphodiester backbone at the 3´ and 5´ sides of the abasic site so that base-free deoxyribose is released. USER (Uracil-Specific Excision Reagent) Enzyme, combines these two enzymatic activities to generate a single nucleotide gap at the location of a uracil residue.
USER (Uracil-Specific Excision
Reagent) Enzyme (1) generates a single nucleotide
gap at the location of a uracil. USER Enzyme is a
mixture of Uracil DNA glycosylase (UDG) and the
DNA glycosylase-lyase Endonuclease VIII. UDG
catalyses the excision of a uracil base, forming an
abasic (apyrimidinic) site while leaving the phosphodiester
backbone intact (2,3). The lyase activity
of Endonuclease VIII breaks the phosphodiester
backbone at the 3´ and 5´ sides of the abasic site
so that base-free deoxyribose is released (4,5).
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.
Product Source
The two component proteins are purified separately from E. coli K-12 strains containing plasmids encoding Endonuclease VIII and Uracil-DNA Glycosylase.
This product is related to the following categories:
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
Cavaleiro, A.M., Kim, S.H., Seppälä, S., Nielsen, M.T., Nørholm, M.H.H. (2015) Accurate DNA Assembly and Genome Engineering with Optimied Uracil Excision Cloning ACS Synth Biol; 4 (9), 1042-1046. DOI: 10.1021/acssynbio.5b00113
Salomonsen B., Mortensen U.H., Halkier B.A. (2014) USER-Derived Cloning Methods and Their Primer Design In: Valla S., Lale R. (eds) DNA Cloning and Assembly Methods Methods in Molecular Biology (Methods and Protocols); 116, 59-72. DOI: 10.1007/978-1-62703-764-8_5
Wang S, Li W, Wang S, Hu B (2014) Rapid and Efficient Assembly of Transcription Activator-Like Effector Genes by USER Cloning J Genet Genomics; 41(6), 339-47. PubMedID: 24976123, DOI: 10.1016/j.jgg.2014.05.002
Lund, A.M., Kildegaard, H.F., Petersen, M.B.K., Rank, J., Hansen, B.G., Andersen, M.R., et al. (2014) A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering PLoS One; 9 (5), e96693. DOI: 10.1371/journal.pone.0096693
Lund AM, Kildegaard HF, Petersen MB, Rank J, Hansen BG, Andersen MR, Mortensen UH (2014) A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering PLoS One; 9(5), e96693. PubMedID: 24879460, DOI: 10.1371/journal.pone.0096693
Villiers BR, Stein V, Hollfelder F (2010) USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction Protein Eng Des Sel; 23(1), 1-8. PubMedID: 19897542, DOI: 10.1093/protein/gzp063
Nour-Eldin H.H., Geu-Flores F., Halkier B.A. (2010) USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories In: Fett-Neto A. (eds) Plant Secondary Metabolism Engineering. Methods in Molecular Biology (Methods and Protocols); 643, 185-200. DOI: 10.1007/978-1-60761-723-5_13
Publications
Thuronyi, B.W., Koblan, L.W., Levy, J.M. et al (2019) Continuous evolution of base editors with expanded target compatibility and improved activity Nat Biotechnol; 37, 1070-1079. PubMedID: 31332326, DOI: 10.1038/s41587-019-0193-0
Quality, Safety & Legal
Quality Control Assays
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
USER Enzyme has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10233215. All subsequent (higher number) lots will contain rAlbumin.
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]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
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.
