USER™ Enzyme

Description

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.

Product Source

The two component proteins are purified separately from E. coli K-12 strains containing plasmids encoding Endonuclease VIII and Uracil-DNA Glycosylase.

Supplied in: 50 mM KCl, 5 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 175 mg/ml BSA and 50% glycerol.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
CutSmart® Buffer-2010X

Properties and 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 CutSmart® Buffer
Incubate at 37°C

1X CutSmart® Buffer:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
pH 7.9 @ 25°C

Storage Temperature

-20°C

Storage Conditions

10 mM Tris-HCl
5 mM NaCl
1 mM DTT
0.1 mM EDTA
175 μg/ml BSA
50% Glycerol
50 mM KCl
pH 7.4 @ 25°C

Heat Inactivation

No

Notes

  1. 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). For further information, see the USER Friendly Cloning Kit manual (NEB #E5500).
  2. Incubate at 37°C.

References

  1. Bitinaite, J. unpublished observations.
  2. Lindhal, T., Ljungquist, S., Siegert, W., Nyberg, B. and Sperens, B. (1977). J. Biol. Chem.. 252, 3286-3294.
  3. Lindhal, T. (1982). Annu. Rev. Biochem.. 51, 61-64.
  4. Melamede, R.J., Hatahet, Z., Kow, Y.W., Ide, H. and Wallace, S.S. (1994). Biochemistry. 33, 1255-1264.
  5. Jiang, D., Hatahet, Z., Melamede, R.J., Kow, Y.W. and Wallace, S.S. (1997). J. Biol. Chem.. 272, 32230-32239.

FAQs

  1. Will the USER Enzyme work in CutSmart buffer?

Protocols

  1. Cloning with USER Enzyme

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Selection Charts

Usage Guidelines & Tips

Interactive Tools

Citations

  • 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
  • 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
  • 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

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Functional Test (USER, Transformation Assay):
    A 10 μl reaction in ThermoPol Reaction Buffer containing 20 ng linearized pNEB206A, 100 ng of a 950 bp control PCR product and 1 unit of USER Enzyme was incubated for 15 minutes at 37°C followed by 15 minutes at 25°C. After transformation into ER2267 chemically-competent cells > 95% of colonies contained recombinant plasmid.

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.