NruI

Cat. No.: L398135

GRADE & PURITY BioReagent, PCR Reagent, for DNA synthesis, Suitable for molecular biology, EnzymoPure™, For In Vitro Transcription, for DNA and RNA applications, 20 U/μl

Overview

Storage buffer: 10mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 200µg/ml Recombinant Albumin, 50% Glycerol, pH 7.4

NruI can recognize the TCG/CGA palindromic sequence, and form a flat terminal after enzyme digestion. The isozymes of NRUI include Bsp68I, BtuMI and RruI. Recombinant albumin was added to both NruI and 10×Cut Reaction Buffer to ensure the safety and stability of the product.

Product composition

L398135	Component	1 KU	5 KU	Storage
L398135A	NruI	50 μL	250 μL	-20°C. Avoid freeze/thaw cycle
L398135B	10×Cut Reaction Buffer	400 μL	2 mL	-20°C. Avoid freeze/thaw cycle

Source
E.coli

Restriction Enzyme CuttingSite
5’…TCG↓CGA…3’
3’…AGC↑GCT…5’ 

Enzyme Activity Definition

In a total reaction system of 50 μl, the amount of enzyme required for enzyme digestion of 1 μg λDNA within 1 h at 37℃ was defined as one active single site (U).
Protocol
(1) Configure the reaction system, as shown in the following table

Component	volume
RNase-free Water	To 50 μl
10×Cut Reaction Buffer	5 μl
DNA	1 μg
NruI	1 μl*

(2) Gently mix, do not oscillate the vortex, and then instantaneous centrifugation.
(3) Incubation at 37°C for 30 min.
Note: *Nrul is finally added to the system, generally 50 μl reaction system to add 1μl of Nrul, can also be adjusted according to the experiment, it is recommended that the amount of enzyme added does not exceed 10% of the total system to avoid star activity.
Cautions
(1) Methylation sensitivity of Nrul:
Dam methylation: Methylation and cleavage sites may overlap resulting in impaired cleavage
Dcm methylation: insensitive
CpG methylation: Blocking cleavage
(2) Before the Meche reaction, try to ensure that the DNA does not contain impurities such as phenol, chloroform, alcohol, EDTA, so as not to affect the effect of enzyme digestion.
(3) This product is for scientific research only and shall not be used for other purposes.

Specifications

Product Name

NruI

Synonyms

NruI | Bsp68I | BtuMI | RruI

Grade

BioReagent, for DNA and RNA applications, for DNA synthesis, For In Vitro Transcription, PCR Reagent, Suitable for molecular biology, EnzymoPure™

Specifications & Purity

BioReagent, PCR Reagent, for DNA synthesis, Suitable for molecular biology, EnzymoPure™, For In Vitro Transcription, for DNA and RNA applications, 20 U/μl

Molecule Type

Enzyme

Product Line

Biological Products

Storage and Shipping

Concentration

20 U/μl

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

Stability And Storage

Store at -20℃ long term (12 months). Upon delivery aliquot. Avoid freeze/thaw cycle.

Application Protocols

Literature-based example protocol for NruI restriction digestion (adjust based on CoA and your lab’s SOPs):

• Goal: Digest 1 µg plasmid DNA to completion for cloning/analysis.
• Materials: NruI enzyme, 10× restriction buffer compatible with NruI, nuclease-free water, DNA sample, optional BSA, ice, microcentrifuge tubes.
• Steps:
  1. Thaw buffer and DNA on ice. Keep enzyme on ice at all times. Briefly vortex buffer and water; spin down.
  2. Assemble on ice: water → 10× buffer (to 1× final) → DNA (1 µg) → optional BSA (per buffer recommendation) → NruI (add last). Keep final glycerol ≤5–10% v/v to avoid star activity.
  3. Mix gently by pipetting; quick spin-down.
  4. Incubate at the recommended temperature (commonly 37°C for Type II enzymes) for 15–60 min (analytical) or up to 2–4 h (preparative). Avoid prolonged overdigestion unless validated.
  5. Stop reaction by heat inactivation if supported for NruI (check CoA) or purify DNA using a spin column/bead cleanup.
  6. Analyze an aliquot by agarose gel electrophoresis to confirm expected band sizes.
• Notes:
  • For blunt-end ligation: After digestion, consider dephosphorylation of vector ends if preventing recircularization, and use higher ligase concentration or longer ligation time.
  • For double digests: Use a buffer compatible with both enzymes or perform sequential digests with cleanup between steps.

Item-specific unit definition, recommended buffer formulation, and heat-inactivation conditions: Not specified for this item; refer to CoA/Spec Sheet.

Biological Roles

NruI is derived from bacterial restriction–modification (R–M) systems that protect host genomes from invading phage DNA.

• Role in native organisms (literature/general):
  • Type II restriction endonucleases recognize specific palindromic DNA sequences and cleave foreign DNA. The cognate methyltransferase modifies host DNA at the same recognition sequence, preventing self-cleavage.
• Mechanism overview (literature/general):
  • Binds DNA as a dimer at the target site, positions catalytic residues to hydrolyze phosphodiester bonds in the presence of Mg2+, and yields a precise, blunt-ended break at TCG^CGA for NruI.
• Relevance to research:
  • Provides a precise tool to probe DNA–protein interactions, chromosomal organization (restriction mapping), and construction of engineered DNA for synthetic biology.

Note: This product is intended for research use only; it is not intended for diagnostic or therapeutic use.

Buffer Applications

This product is an active enzyme rather than a buffering reagent. It is used within restriction digestion buffers tailored to the enzyme’s requirements.

• Typical digestion buffer composition (literature/general):
  • 1× Tris-based buffer at pH ~7.5–8.0, MgCl2 (typically 10 mM), salt (e.g., NaCl or KCl), and often BSA as a stabilizer. Some commercial systems provide a single “universal” buffer compatible with many enzymes.
• Practical notes:
  • Use nuclease-free water and certified molecular biology-grade reagents.
  • Final glycerol concentration in the reaction should generally be kept ≤5–10% to avoid altered specificity (star activity).
  • If conducting double digests, select a buffer that provides acceptable activity for both enzymes or perform sequential digests with cleanup in between.

Item-specific buffer recommendation, composition of storage buffer, and activity profile across buffers: Not specified for this item; refer to CoA/Spec Sheet.

Green Alternatives

Restriction endonucleases are aqueous, low-volatility reagents. Environmental impact arises mainly from cold-chain transport, plastic consumables, and buffer components.

• Greener practice suggestions (literature/general):
  • Plan digests to minimize repeat reactions and plastic use; opt for multi-well formats and reusable cold blocks instead of single-use ice packs when practical.
  • Use concentrated stocks to reduce packaging and transport volume where feasible (subject to activity and stability constraints; item-specific details not specified).
  • Choose buffers free of hazardous additives and prepare with ultrapure water; avoid unnecessary solvent additives.
  • Consolidate shipping with other cold-chain reagents to reduce packaging waste. This product is shipped cold (Product Data: Ice chest + Ice pads).
  • Consider room-temperature-stable formulations if available for your workflow; trade-off is often reduced shelf-life or altered performance (general note; not specific to this item).

Trade-offs: Preserving enzyme activity typically requires −20°C storage and cold shipment, which increases energy and packaging usage. Aliquoting to reduce waste and extending in-use life can offset repeated shipments.

Pharmaceutical Uses

Not typically applicable. Restriction endonucleases like NruI are tools for laboratory DNA manipulation rather than formulation excipients or process aids in finished pharmaceutical products. When used in bioprocess development or gene-construct preparation, they remain research/manufacturing intermediates and are not present in final drug products. No pharmacopeial monograph is associated with this enzyme. For any GMP-related use, obtain full quality documentation and perform suitability assessments.

Physical Properties

This product is a protein enzyme preparation; small-molecule physical constants do not apply.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Concentration/formulation: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight (protein): Not specified for this item; refer to CoA/Spec Sheet. (Literature note: many Type II restriction endonucleases are ~25–40 kDa per monomer.)
• pI, extinction coefficient: Not specified for this item; refer to CoA/Spec Sheet.
• Solubility (literature/general): Readily soluble in aqueous biological buffers; typically supplied in glycerol-containing storage buffer to maintain activity at subzero storage temperatures.
• Stability (literature/general): Activity is preserved at −20°C for extended periods when stored in stabilized buffers and protected from repeated freeze–thaw cycles. Activity decreases with prolonged incubation above 25–30°C or in the absence of required divalent cations.
• Cofactor dependence (literature/general): Requires Mg2+ for catalysis; activity is sensitive to ionic strength and pH, with typical optima near neutral to slightly alkaline pH.

Quality & Grades

This item is offered for demanding molecular biology workflows.

• Declared item grades (from Product Data):
  • BioReagent; PCR Reagent; For DNA synthesis; Suitable for molecular biology; EnzymoPure™; For In Vitro Transcription; for DNA and RNA applications.
• Practical implications:
  • BioReagent / Suitable for molecular biology: Typically indicates sourcing and handling compatible with nucleic acid work and low bioburden (general description; not a guarantee of specific contaminants unless stated on CoA/SDS).
  • PCR Reagent / for DNA and RNA applications: Generally suggests very low levels of contaminating DNases/RNases and inhibitors (literature convention). Item-specific nuclease testing results: Not specified for this item; refer to CoA/Spec Sheet.
  • EnzymoPure™: Branding that implies high enzymatic purity and functional performance. Exact specifications (specific activity, unit definition, host-derived impurity levels) are Not specified for this item; refer to CoA/Spec Sheet.
  • For In Vitro Transcription / For DNA synthesis: Indicates compatibility with enzymatic workflows that require clean DNA preparations and sequence-specific processing. No claim is made about therapeutic-grade status.
• UV cutoff, metal content, nuclease/endonuclease background, and residual host nucleic acids: Not specified for this item; refer to CoA/Spec Sheet.
• Documentation: Request the Certificate of Analysis for lot-specific activity (U/µL), recommended buffer, QC assays (e.g., self-digestion, nicking), and stability testing.

Reaction & Applications

NruI is a sequence-specific DNA endonuclease for precise double-strand cleavage, central to cloning and synthetic biology.

• Core applications (literature/general):
  • Restriction digest of plasmids/genomic DNA at 5'-TCGCGA-3' sites, producing blunt ends (TCG^CGA).
  • Vector/insert preparation for blunt-end ligation, adapter/barcode ligation after end-repair, and construction of libraries for NGS.
  • Restriction mapping and verification of constructs via diagnostic digests.
  • Generation of defined DNA fragments for in vitro transcription templates or gene assembly workflows.
• Methylation sensitivity (literature/general): Cleavage at sites containing 5-methylcytosine within CpG may be inhibited; dam/dcm methylation in E. coli can also affect some recognition sites. Validate with methylation state of source DNA.
• Practical tips (literature/general):
  • Use nuclease-free water, keep enzyme on ice, and add enzyme last after buffer and DNA to minimize local high salt/denaturant exposure.
  • Avoid excessive glycerol in the reaction (>5–10% v/v) to limit star activity and maintain ionic strength.
  • For blunt-end ligations, use higher DNA concentrations and increased ligase or longer ligation times compared with sticky-end ligations.
  • Confirm digestion by agarose gel electrophoresis; include an undigested control.
• Compatibility: Double-digests are feasible when buffer/temperature optima overlap; otherwise, perform sequential digests with cleanup or heat-inactivate enzymes if applicable (check enzyme-specific heat inactivation; not specified for this item).

Reaction Conditions

General guidance for restriction digestion with NruI (literature-based; adjust per CoA/SDS and your lab SOPs):

• Reaction setup (typical):
  • DNA: up to 1 µg per 50 µL reaction is a common benchmark for routine analysis; higher loads may require proportionally more enzyme or longer incubation.
  • Buffer: 1× restriction buffer compatible with NruI (Tris-HCl pH ~7.5–8.0, Mg2+ present). Include BSA if recommended.
  • Enzyme: Many suppliers define 1 Unit as the amount that completely digests 1 µg of standard DNA in 1 h at the optimal temperature. Use 1–10 U per µg DNA depending on application stringency.
  • Temperature: Often 37°C for Type II enzymes; verify for NruI in the CoA. Some enzymes tolerate 25–50°C ranges with altered rates.
  • Time: 15–60 min for analytical digests; 1–4 h (or overnight under controlled conditions) for preparative digests.
• Controls and verification:
  • Include an undigested DNA control and, if needed, a methylation control to assess potential inhibition.
  • Analyze products by agarose gel electrophoresis; for blunt-end ligations, confirm end integrity.
• Avoiding star activity (literature/general):
  • Keep glycerol ≤5–10% v/v, maintain recommended ionic strength and pH, avoid organic solvents/detergents, and do not overincubate with excessive enzyme.
• Heat inactivation/cleanup:
  • Some restriction enzymes can be heat-inactivated (e.g., 65–80°C for 10–20 min); check NruI-specific guidance in the CoA. If not heat-inactivatable, purify DNA by spin column or phenol-free cleanup.

Item-specific optimal temperature, buffer brand/formulation, and unit activity: Not specified for this item; refer to CoA/Spec Sheet.

Safety & Handling

For research use only. Handle using good laboratory hygiene and aseptic technique to prevent contamination of biological samples.

• GHS/CLP classification: Not specified for this item; refer to SDS. Protein enzymes for molecular biology are generally not classified as hazardous mixtures, but authoritative classification is provided in the SDS.
• Signal word / hazard statements / pictograms: Not specified for this item; refer to SDS.
• Primary hazards (general):
  • Low acute toxicity expected; avoid inhalation of aerosols and contact with eyes/skin.
  • May contain preservatives or stabilizers (e.g., salts, reducing agents) depending on formulation (not specified for this item); check SDS for sensitization or irritation risks.
• PPE recommendations (good practice):
  • Wear lab coat, disposable nitrile gloves, and safety glasses.
  • Work in a clean area; keep vials on ice during use to preserve activity.
• Incompatibilities and conditions to avoid (general):
  • Avoid metal chelators (EDTA, EGTA) in the reaction mix at inhibitory levels; these can strip Mg2+ required for activity.
  • Avoid proteases and extreme pH; enzyme is denatured by heat (>65°C sustained) and by organic solvents.
• First aid (overview; defer to SDS):
  • Skin/eye contact: Rinse with water; remove contaminated clothing. Seek medical attention if irritation persists.
  • Ingestion/inhalation: Rinse mouth, move to fresh air; obtain medical advice if unwell.
• Spill/cleanup: Absorb aqueous spills with paper towels; decontaminate surfaces with 70% ethanol or appropriate disinfectant. Dispose according to local regulations.

Solvent Selection

Not typically applicable. NruI is a water-soluble protein supplied and used in aqueous biological buffers. Use nuclease-free water and the recommended reaction/storage buffers. Avoid organic solvents and strong denaturants, which inactivate the enzyme.

Storage & Reconstitution

• Storage (from Product Data): Store at −20°C. Avoid repeated freezing and thawing. Shipped in an ice chest with ice pads to maintain cold chain.
• Handling practices (literature/general):
  • Keep enzyme on ice during setup. Return immediately to −20°C after use.
  • Aliquot upon first thaw to minimize freeze–thaw cycles and activity loss.
  • Use low-retention, nuclease-free tubes and tips to avoid adsorption and contamination.
  • Do not store near proteases or strong oxidants; avoid microbial contamination by using sterile technique.
• Reconstitution: Not specified for this item; refer to CoA/Spec Sheet. Many restriction enzymes are supplied ready-to-use in stabilized aqueous buffer; if lyophilized (not specified here), reconstitute with nuclease-free buffer per instructions.
• Stability: Item-specific shelf life and activity retention profile are Not specified for this item; refer to CoA/Spec Sheet. As a general guideline, sustained storage at −20°C in a stabilizing buffer preserves activity for months; avoid temperature excursions.
• Transport: Maintain cold chain upon receipt; verify integrity of cold packs and absence of accidental warming. If exposure is suspected, perform a small test digest to confirm activity before critical experiments.

Structure & Identity

NruI is a Type II restriction endonuclease used for sequence-specific cleavage of double-stranded DNA in molecular biology workflows.

• Item-specific identifiers:
  • CAS: Not specified for this item; refer to CoA/Spec Sheet.
  • CID: Not specified for this item; refer to CoA/Spec Sheet.
  • InChIKey: Not applicable (protein enzyme); not assigned.
  • SMILES: Not applicable for macromolecular proteins.
  • Molecular Formula: Not applicable in empirical small-molecule notation for enzymes.
  • Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.
• Structural/biochemical features (literature/general):
  • Enzyme class: Type II restriction endonuclease (EC 3.1.21.4), typically Mg2+-dependent and sequence-specific.
  • Recognition sequence and cleavage pattern: NruI recognizes the palindromic hexamer TCGCGA and cleaves to generate blunt ends at TCG^CGA (literature).
  • Typical quaternary structure: Many Type II restriction enzymes are homodimers that bind palindromic sites; NruI is reported to act as a dimer at its target site (literature).
  • Reaction product: Blunt-ended double-strand DNA breaks suitable for blunt-end ligation, end-repair, or adapter ligation workflows (literature).
• 2D/sequence description in words (literature/general):
  • Binds a six-base palindromic target (5'-TCGCGA-3'/3'-AGCGCT-5'), positions the scissile phosphodiester bonds symmetrically across the dyad axis, and introduces a double-strand break to yield flush (0-nt overhang) ends.

Synthetic Utility

In DNA engineering, NruI functions as a precision “reagent” for constructing and editing nucleic acid sequences.

• Retrosynthetic planning with restriction sites (literature/general):
  • Introduce or remove the TCGCGA site via PCR primers or gene synthesis to enable modular cloning steps.
  • Use NruI’s blunt ends to generate fragments compatible with a wide range of blunt-end workflows (e.g., ligation into EcoRV-, SmaI-, or PmeI-linearized vectors after end-polishing), reducing sequence constraints.
• Assembly workflows:
  • Combine with other restriction enzymes to generate non-compatible ends that enforce insert orientation after end-repair/adaptor strategies.
  • Create linear templates for in vitro transcription by releasing promoter–gene cassettes at a defined distance from the terminator.
• Quality control:
  • Diagnostic digestion to verify plasmid architecture and confirm presence/absence of engineered NruI sites.
• Design considerations:
  • Blunt-end ligation lacks inherent directionality; incorporate additional sequence features (secondary restriction site or asymmetric adapters) to enforce orientation.
  • Check for methylation sensitivity of cloning hosts (dam/dcm or CpG methylation) that may block cleavage at TCGCGA.

These are general literature-based practices; adapt to your construct and obtain lot-specific activity data from the CoA.

Target Specificity

Not applicable to antibodies or ligand-binding probes. As a restriction endonuclease, NruI’s specificity is enzymatic: it recognizes the DNA sequence 5'-TCGCGA-3' and cleaves to produce blunt ends (literature). No monoclonal clone, isotype, or species reactivity applies.

Frequently Asked Questions

What does PCR Reagent mean?

PCR Reagent indicates that the product has been processed and tested for PCR, sequencing and nucleic acid amplification. Typical specifications include low or undetectable nuclease/protease activity, low endotoxin, controlled microbial limits and high lot-to-lot reproducibility.

What does BioReagent mean?

BioReagent indicates that the product has been processed and tested for biological and biochemical applications. Specifications may include endotoxin limits, microbial controls, sterility, low DNase/RNase/protease activity, and verified biological activity where relevant.

How should this product be stored?

Store at ?20 °C. Avoid repeated freeze–thaw cycles. Aliquot into single-use volumes before freezing.

How is this product shipped?

This product ships in an insulated container with ice pads. Unpack on arrival and transfer it to the storage condition stated above.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.