Product Class: Other

Thermolabile USER® II Enzyme
neb31 cloned at NEB recombinant 37 65 Heat

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.


Product Introduction

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

Applications of Thermolabile USER II include:

  • Directional RNA Seq
  • NEBNext adaptor cleavage
  • USER cloning

Figure 1: Thermolabile USER II Enzyme

Mechanism
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.


Catalog # Size Concentration
M5508S 50.0 units 1000 units/ml
M5508L 250.0 units 1000 units/ml

Product Information

Description

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 Enyzme and Thermolabile USER II Enzyme for Adaptor Cleavage are Equivalent
Adaptor Cleavage
(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:
DNA Repair Enzymes and Structure-specific Endonucleases Products,
This product can be used in the following applications:
Applications of USER® and Thermolabile USER II Enzymes,
USER® Cloning,
DNA Assembly and Cloning,
High-throughput cloning and automation solutions

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 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)

Activity in NEBuffers

rCutSmart™ Buffer: 100%
NEBuffer™ r1.1: 100%
NEBuffer™ r2.1: 100%
NEBuffer™ r3.1: 100%

Storage Buffer

15 mM Tris-HCl
35 mM NaCl
25 mM KCl
1 mM DTT
0.1 mM EDTA
100 µg/ml BSA
50% Glycerol
pH 7.5 @ 25°C

Heat Inactivation

65°C for 10 minutes

Product Notes

  1. Thermolabile USER II 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).

References

  1. Lindhal, T. et al. (1977). J. Biol. Chem. 252, 3286-3294.
  2. Lindhal, T. (1982). Annu. Rev. Biochem. 51, 61-64.

Protocols, Manuals & Usage

Protocols

  1. Thermolabile USER® II Enzyme Applications in Next Generation Sequencing Library Preparation (M5508)
  2. Thermolabile USER® II Enzyme Standard Protocol (M5508)

FAQs & Troubleshooting

FAQs

  1. What is the activity of Thermolabile USER II Enzyme in other buffers?
  2. Can Thermolabile USER Enzyme be heat inactivated?
  3. How is Thermolabile USER® II Enzyme different than USER Enzyme?
  4. Will Thermolabile USER II Enzyme (NEB #M5508) remove uracil from ssDNA and dsDNA?
  5. What applications are Thermolabile USER® II Enzyme best suited for?
  6. During an NEBNext DNA library prep, which enzyme cleaves NEBNext DNA loop adaptors and uracil-containing single stranded loops?
  7. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

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.

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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