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 (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:
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)
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