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USER® Enzyme

Catalog # Concentration Size List Price Quantity Your Price
M5505L 1000 units/ml 250 units $489.00
$440.10
M5505S 1000 units/ml 50 units $120.00
$108.00
Catalog # Size List Price Your Price
M5505L 250 units $489.00
$440.10
M5505S 50 units $120.00
$108.00
Catalog #
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

USER (Uracil-Specific Excision Reagent) Enzyme (1) generates a single nucleotide gap at the location of a uracil residue.

Applications of USER include:

  • Directional RNA Seq
  • NEBNext adaptor cleavage
  • USER cloning

 

Figure 1: USER Enzyme

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.


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    What are endonucleases and their applications?
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).

To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.

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.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  USER® Enzyme M5505SVIAL -20 1 x 0.05 ml 1,000 units/ml
  rCutSmart™ Buffer B6004SVIAL -20 1 x 1.25 ml 10 X
  USER® Enzyme M5505LVIAL -20 1 x 0.25 ml 1,000 units/ml
  rCutSmart™ Buffer B6004SVIAL -20 1 x 1.25 ml 10 X

Properties & Usage

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

Companion Products
Notes
  • USER Enzyme is active in all commercial PCR buffers tested. It also has 100% activity in TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA).
  • Incubate at 37°C.
References
  • Bitinaite, J. unpublished observations.
  • Lindhal, T., Ljungquist, S., Siegert, W., Nyberg, B. and Sperens, B. (1977). J. Biol. Chem.. 252, 3286-3294.
  • Lindhal, T. (1982). Annu. Rev. Biochem.. 51, 61-64.
  • Melamede, R.J., Hatahet, Z., Kow, Y.W., Ide, H. and Wallace, S.S. (1994). Biochemistry. 33, 1255-1264.
  • Jiang, D., Hatahet, Z., Melamede, R.J., Kow, Y.W. and Wallace, S.S. (1997). J. Biol. Chem.. 272, 32230-32239.
Citations
Additional Citations
  • 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 Control Assay
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.
Change Notifications

USER Enzyme has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10233215. All subsequent (higher number) lots will contain rAlbumin.

Specifications
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]
Certificate of Analysis
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]
Safety Data Sheets
Legal And Disclaimer

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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.

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