Product Class: Other

USER® Enzyme
Gray property icon for rCutSmart Gray property icon for cloned at NEB recombinant Gray property icon for incubation temperature 37° Gray property icon for heat inactivation no

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.

Product Introduction

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

 

USER Enzyme

Workflow showing mechanism of USER enzyme as compared to Uracil DNA Glycosylase (UDG) and Endonuclease VIII
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.


Bioz Badge Exists : True
Catalog # Size Concentration
M5505S 50 units 1000 units/ml
M5505L 250 units 1000 units/ml

Product Information

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.

Figure 1: 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

Comparison of mechanisms of USER Enzyme, USER II Enzyme and USER III Enzyme

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:
DNA Repair Enzymes and Structure-specific Endonucleases
This product can be used in the following applications:
Applications of USER® and Thermolabile USER II Enzymes,
USER® Cloning,
DNA Assembly and Cloning

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

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

Product 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).
  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.

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. Will the USER Enzyme work in rCutSmart buffer?
  2. Where can I find information or a protocol for cloning with USER® Enzyme?
  3. During an NEBNext DNA library prep, which enzyme cleaves NEBNext DNA loop adaptors and uracil-containing single stranded loops?
  4. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?
  5. What is the difference between the suite of USER® enzymes (#M5505, #M5508, #M5509) and Thermostable Endonuclease Q (#M0701)?
  6. Do any of the USER enzymes leave ligatable ends when cleaving at a Uracil?
  7. How close to the ends of DNA can USER Enzyme (NEB #M5505) cleave?

Quality, Safety & Legal

Quality Assurance Statement

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.

Specification 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 DataSheets

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

Legal and Disclaimers

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.

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