Product Class: Restriction Endonuclease

WarmStart® Nt.BstNBI
neb31 cloned at NEB recombinant dil_A 55 80 Heat hotstart


Catalog #R0725

Isoschizomers | Single Letter Code

Product Introduction

  • This is a nicking endonuclease
  • WarmStart technology inhibits enzyme activity below 40°C
  • Generates DNA molecules that are “nicked,” rather than cleaved
  • Allows for room temperature setup, facilitating SDA
  • Nicking Enzyme Cut Site: GAGTC (4/none)

Product Information

Description

WarmStart Nt.BstNBI is a site specific nicking endonuclease cloned from Bacillus Stereothermophilus. It is formulated with a reversibly-bound aptamer, which inhibits its nicking activity at temperatures below 40°C. WarmStart Nt.BstNBI cleaves only one strand of DNA on a double-stranded DNA substrate. The nicking endonuclease catalyzes a single strand break 4 bases beyond the 3′ side of the recognition sequence.

Highlights

  • Nt.BstNBI catalyzes a single-stranded nick 3′ of its recognition sequence. WarmStart functionality is achieved by formulation with a DNA aptamer that inhibits enzyme activity below 40°C
Figure 1: No detectable activity of WarmStart Nt.BstNBI at 25 °C



A) Nicking activity on phage T7 DNA. 1 μg T7 phage DNA was incubated with various amounts of WarmStart Nt.BstNBI (NEB #R0725) and Nt.BstNBI (Non-WarmStart, NEB #R0607) in a 50 μl reaction in NEBuffer r3.1 for 1 hour at 25°C or 55°C. 8 units of enzyme was added and a 2-fold serial dilution was performed. As indicated with “+”, full activity is observed for both enzymes at the permissive temp (55°C) but not at the restrictive temp (25 °C) for the WarmStart version.

B) Nicking activity on a fluorescently labeled DNA duplex. 2 pmol 48-bp DNA duplex containing an internal recognition site for Nt.BstNBI was incubated with various amounts of WarmStart Nt.BstNBI (NEB #R0725) and Nt.BstNBI (Non-WarmStart, NEB #R0607) in a 20 μl reaction with NEBuffer r3.1 for 30 min at 25°C or 55°C. 10 units of enzyme was added and a 2-fold serial dilution was performed. The reactions were quenched in 20 mM EDTA, 0.1% Tween 20 and then diluted with water to the final concentration of FAM-labeled DNA duplex at 1 nM. The reactions were analyzed by capillary electrophoresis (CE) fragment analysis on an Applied Biosystems 3730xl Genetic Analyzer (96 capillary array). % Product was determined as the area of the product peak divided by the total area of all peaks in the FAM channel. The average and the standard deviation of % product (Y-axis) plotted with log phase of nicking enzyme units (X-axis) was taken from triplicate reactions.

Conclusion: The WarmStart version exhibits similar activity to the regular version at 55°C but has no detectable activity at 25°C.



Figure 2: Reaction temperature profile of WarmStart Nt.BstNBI 



Nicking activity of WarmStart Nt.BstNBI (NEB #R0725) and Nt.BstNBI (Non-WarmStart NEB #R0607) on a fluorescently-labeled DNA duplex was measured at various temperatures (25°C to 80°C, 5°C intervals). Reactions containing 0.125 unit WarmStart or non- WarmStart Nt.BstNBI, 100 nM DNA duplex (5’ 6-FAM labeled) in 20 μl NEBuffer r3.1 were incubated for 30 min at various temperatures. The average and the standard deviation of % product (Y-axis) plotted with temperatures (X-axis) was taken from triplicate reactions.

Conclusion: WarmStart Nt.BstNBI shows less than 10% activity below 40°C and is undetectable below 30°C, thereby enabling reaction setup at room temperature with no unintended conversion of substrate. 



Figure 3: WarmStart Nt.BstNBI facilitates SDA by allowing room temperature setup 



Identical Strand Displacement Amplification (SDA) reactions were run either after incubation at 25°C for 30 minutes (to mimic room temperature reaction setup) or immediately after setup on ice, using WarmStart Nt.BstNBI (NEB #R0725) or Nt.BstNBI (Non-WarmStart, NEB #R0607). Reactions were performed in 25 μl of 1X Isothermal Amplification Buffer (NEB #B0537) with 0.5 μl LAMP Fluorescent Dye (NEB #B1700), 0.4 mM dNTPs (NEB #N0447), 10 ng of template DNA (HeLa genomic DNA, 290 copies), 0.5 mM of each primer, 1 μl Bst 2.0® Polymerase (NEB #M0537S) and 1 μl WarmStart Nt.BstNBI or Nt.BstNBI.  Isothermal amplification was performed at 55°C and fluorescence was monitored for 45 minutes. The time differential for the appearance of positive signal in test samples and negative controls is distinguishable only for reactions  set up on ice (both versions) or at room temperature (WarmStart version only). The variation between technical replicates was low. 

Conclusion: Only WarmStart Nt.BstNBI enabled successful reaction setup at room temp for SDA reactions targeting a hBRCA1 amplicon in genomic DNA. 

 



Product Source

An E. coli strain that carries the cloned Nt.BstNBI gene from Bacillus stereothermophilus 33M (Z. Chen).
This product is related to the following categories:
Nicking Endonucleases Products,
Restriction Endonucleases: N-O Products,
Restriction Endonucleases Products,
This product can be used in the following applications:
Strand Displacement Amplification & Nicking Enzyme

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 digest 1 µg T7 DNA in NEBuffer r3.1 in 1 hour at 55°C in a total reaction volume of 50 µl.

Reaction Conditions

1X NEBuffer™ r3.1
Incubate at 55°C

1X NEBuffer™ r3.1
100 mM NaCl
50 mM Tris-HCl
10 mM MgCl2
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)

Usage Concentration

10X

Activity in NEBuffers

NEBuffer™ r1.1: 0%
NEBuffer™ r2.1: 10%
NEBuffer™ r3.1: 100%
rCutSmart™ Buffer: 25%

Diluent Compatibility

Storage Buffer

10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
200 µg/ml Recombinant Albumin
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

80°C for 20 minutes

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive

Activity at Temperature

@37°C: 0%

Features

  • WarmStart technology allows for room temperature setup, making applications such as Strand Displacement Amplification (SDA) user friendly.
  • WarmStart technology inhibits the unintentional reaction below the permissive reaction temperature, which makes experiments consistent and reproducible.

Product Notes

  1. Not sensitive to CpG, dcm, or dam methylation.
  2. The optimal reaction temperature range is 50-60°C. Less than 10% activity is observed when the temperature is below 40°C.
  3. When using WarmStart Nt.BstNBI with Bst 2.0 in strand displacement amplification, specific reaction conditions will vary. For best results, use 1X Isothermal Amplification Buffer.

References

  1. Song, Q. et al. (2010). Anal.Chem. [Epub ahead of print].
  2. Zhang, P. et al. (2010). ProteinExpr. Purif. 69, 226-234. [Epub 2009 Sep 9].
  3. Rohloff, J.C., et al. (2014). Mol Ther Nucleic Acids. 3, e201.
  4. Gelinas, A.D., et al. (2016). Curr Opin Struct Biol. 36, 122–132.

Protocols, Manuals & Usage

Protocols

  1. Standard Protocol for WarmStart® Nt.BstNBI (NEB #R0725)
  2. Strand Displacement Amplification (SDA) Protocol using WarmStart® Nt.BstNBI (NEB #R0725)

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1.  What advantages does WarmStart® Nt.BstNBI provide?
  2. What is Strand Displacement Amplification (SDA)?
  3. When is star activity a concern?
  4. Is Gel Loading Dye, Purple (6X) or Gel Loading Dye, Purple (6X), no SDS compatible with other DNA binding dyes such as SYBR® and GelRed™ during gel electrophoresis?
  5. Which NEB restriction enzymes are supplied with Gel Loading Dye, Purple (6X)?

Troubleshooting

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.

Licenses

Nucleic acid-based aptamers for use with nicking enzymes are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the WarmStart® Nt.BstNBI for Research Use Only (RUO). Commercial use of this product may require a license from New England Biolabs, Inc. For additional information or to inquire about commercial use, please contact busdev@neb.com.

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