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NEBNext® Enzymatic 5hmC-seq Conversion Module

Catalog # Concentration Size List Price Quantity Your Price
E3365L 96 reactions $654.00
$588.60
E3365S 24 reactions $173.00
$155.70
Catalog # Size List Price Your Price
E3365L 96 reactions $654.00
$588.60
E3365S 24 reactions $173.00
$155.70
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

NEBNext® Enzymatic 5hmC-seq (E5hmC-seq™) is a new method for the specific detection of 5hmC at the single base level.

The enzymatic conversion workflow minimizes damage to DNA and allows discrimination of 5hmC from both cytosine and 5mC

For E5hmC-seq in Illumina® sequencing workflows, we recommend use of the NEBNext Enzymatic 5hmC-seq Kit (NEB #E3350) instead of this module. The kit includes the E5hmC-seq Adaptor, as well as the high-efficiency NEBNext Ultra™ II library prep reagents, and these components are not included in the Conversion Module.

  • High-sensitivity specific detection of 5hmC
  • 0.1 – 200 ng input range
  • High efficiency conversion
  • Minimal DNA damage
  • E5hmC-seq kit including library prep reagents also available (NEB #E3350)

The NEBNext E5hmC-seq Conversion Module provides a high-performance enzyme-based method for detection of 5hmC at the single base level. In the E5hmC-seq two-step conversion workflow, T4-BGT glucosylates 5hmC, providing protection from deamination by APOBEC in the next step. T4-BGT does not protect 5mC or unmethylated cytosines, which are deaminated by APOBEC to uracils.

Note that for Illumina sequencing workflows, the NEBNext Enzymatic 5hmC-seq Kit, including library prep reagents is available (NEB #E3350)

Figure 1: E5hmC-seq conversion method

E3365 Conversion Module

To enable specific 5hmC detection, 5hmC is first glucosylated using T4-BGT. 5mC and unmodified cytosine are then deaminated by APOBEC to thymine and uracil, respectively, while the protected 5hmC is not converted.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  Control DNA CpG unmethylated Lambda E7123AVIAL -20 1 x 0.024 ml 2 ng/µl
  Control DNA 5hmC T4 E3349AVIAL -20 1 x 0.024 ml 1.5 ng/µl
  NEBNext® Carrier DNA E3351AVIAL -20 1 x 0.024 ml
  Elution Buffer E7124AVIAL -20 1 x 2.1 ml
  NEBNext® Glucosylation Reaction Buffer E3352AVIAL -20 1 x 0.12 ml
  UDP-Glucose E3353AVIAL -20 1 x 0.024 ml
  T4-BGT E3354AVIAL -20 1 x 0.024 ml
  Stop Reagent E7132AVIAL -20 1 x 0.024 ml
  Deamination Reaction Buffer E3356AVIAL -20 1 x 0.096 ml
  Recombinant Albumin E3357AVIAL -20 1 x 0.024 ml
  APOBEC E7133AVIAL -20 1 x 0.024 ml
  Control DNA CpG unmethylated Lambda E7123AAVIAL -20 1 x 0.096 ml 2 ng/µl
  Control DNA 5hmC T4 E3349AAVIAL -20 1 x 0.096 ml 1.5 ng/µl
  NEBNext® Carrier DNA E3351AAVIAL -20 1 x 0.096 ml
  Elution Buffer E7124AAVIAL -20 1 x 8.6 ml
  NEBNext® Glucosylation Reaction Buffer E3352AAVIAL -20 1 x 0.48 ml
  UDP-Glucose E3353AAVIAL -20 1 x 0.096 ml
  T4-BGT E3354AAVIAL -20 1 x 0.096 ml
  Stop Reagent E7132AAVIAL -20 1 x 0.096 ml
  Deamination Reaction Buffer E3356AAVIAL -20 1 x 0.384 ml
  Recombinant Albumin E3357AAVIAL -20 1 x 0.096 ml
  APOBEC E7133AAVIAL -20 1 x 0.096 ml

Properties & Usage

Materials Required but not Supplied

  • If DNA requires fragmentation: NEBNext UltraShear™ (NEB #M7634) or Covaris® instrument and the required tubes or other fragmentation equipment
  • PCR strip tubes or 96-well plates
  • Clean-up beads: SPRIselect™ Reagent Kit (Beckman Coulter®, Inc. #B23317), AMPure® XP beads (Beckman Coulter, Inc. #A63881) or preferred bead manufacturer.
  • Formamide (Sigma #F9037-100 ml), Hi-Di™ Formamide (Thermo Fisher Scientific® #4401457) or 0.1 N NaOH. Formamide is preferred. If using NaOH, please see FAQ on NEB #E3365 FAQ page.
  • 80% Ethanol
  • 1X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA), low TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) or 10 mM Tris-HCl pH 7.5 or 8.0
  • Nuclease-free Water
  • Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515)
  • Metal cooling block, such as Diversified Biotech® (#CHAM-1000)
  • PCR machine
  • Agilent® Bioanalyzer®, TapeStation® or other fragment analyzer and associated consumables


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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
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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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