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NEB/WarmStart? Nt.BstNBI/1,000 units/R0725S
  • NEB/WarmStart? Nt.BstNBI/1,000 units/R0725S

NEB/WarmStart? Nt.BstNBI/1,000 units/R0725S

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貨號: R0725S
品牌: NEB
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    • 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,
      Restriction Endonucleases Products
      This product can be used in the following applications:
      Strand Displacement Amplification & Nicking Enzyme
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