Nt bstnbi nicking endonuclease

Manufactured by New England Biolabs

Sourced in United States

Nt.BstNBI is a nicking endonuclease that recognizes and cleaves the DNA sequence 5'-GAGTCNNNNN^NNNN-3'. It introduces a single-strand nick in the DNA, leaving a 5' overhang.

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Nt.BstNBI (16 citations)

3 protocols using nt bstnbi nicking endonuclease

Enzymatic DNA Modifications and Analysis

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GlaI endonuclease as well as its reaction buffer was supplied by SibEnzyme Ltd (Russia). Nt.BstNBI nicking endonuclease (10 000 U mL^–1), Vent (exo^–) DNA polymerase (2000 U mL^–1), M.SssI CpG methyltransferase (4000 U mL^–1), and S-adenosylmethionine (SAM, 32 mM) were all purchased from New England Biolabs (NEB, USA). TKS Gflex DNA polymerase (1250 U mL^–1), Gflex PCR Buffer, dNTPs (2.5 mM) and the 100 bp DNA ladder marker were obtained from TaKaRa Biotechnology (Dalian, China). 4S Red Plus Nucleic Acid Stain (Life Science, USA) was used for DNA staining in agarose gel electrophoresis. SYBR Green I (20× stock solution in DMSO, 20 mg mL^–1) was purchased from Bio-Vision Biotechnology (Xiamen, China). All of the oligonucleotides (see detailed sequences in Table S1†) used in this work were custom synthesized by TaKaRa Biotechnology (Dalian, China).

Sun Y., Sun Y., Tian W., Liu C., Gao K, & Li Z. (2017). A novel restriction endonuclease GlaI for rapid and highly sensitive detection of DNA methylation coupled with isothermal exponential amplification reaction †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc04975g. Chemical Science, 9(5), 1344-1351.

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EXPAR for Target DNA Detection

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All oligonucleotides were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). The dNTPs mix, Vent (exo-) DNA polymerase (2000 U mL^–1), Nt.BstNBI nicking endonuclease (10 000 U mL^–1), Cas9 nuclease S. pyogenes (1000 nM), HiScribe™ T7 Quick High Yield RNA Synthesis Kit, 10× NEBuffer 3.1 and 10× ThermoPol buffer were purchased from NEB (New England Biolabs, Ipswich, MA, USA). RiboLock RNase inhibitor (40 U μL^–1) and 50× ROX reference dye were purchased from Life Technologies (Carlsbad, CA, USA). EvaGreen® DNA staining dye and GelRed Nucleic Acid Gel Stain dye were purchased from Biotium (Hayward, CA, USA). CFX 96 real-time PCR detection system (Bio-Rad) was used for controlling reaction temperature and measuring real-time fluorescence signal from exponential amplification reaction (EXPAR) for target DNA detection. A NanoDrop 2000 UV-vis spectrophotometer was used for measuring nucleic acid concentration. A Leica TCS SP5 laser scanning confocal microscope was used for fluorescence imaging.

Zhang K., Deng R., Li Y., Zhang L, & Li J. (2016). Cas9 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification †Electronic supplementary information (ESI) available: Sequence information, reaction conditions optimization and Cas9 cleavage analysis in a time dependent manner. See DOI: 10.1039/c6sc01355d. Chemical Science, 7(8), 4951-4957.

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Nuclease-Free Oligonucleotide Preparation

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UltraPure™ Tris-HCI pH 8.0, RNase free EDTA, RNase free MgCl₂, RNase free KCl, Novex™ TBE Running Buffer (5X), 2X TBE-Urea Sample Buffer, Novex™ TBE-Urea Gels, 15%, SYBR^® Gold Nucleic Acid Gel Stain, and SYBR^® Green II RNA Gel Stain were purchased from Thermo Fisher Scientific (Waltham, MA). Nuclease-free water and oligo length standard 10/60 were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Nt.BstNBI nicking endonuclease, Bst 2.0 WarmStart^® DNA Polymerase, 10× ThermoPol I Buffer, dNTPs, BSA, and 100 mM MgSO₄ were purchased from New England Biolabs (Beverly, MA).
Oligonucleotides were ordered from two different sources to avoid trigger contamination in templates. Desalted amplification templates were purchased from Integrated DNA Technologies (Coralville, IA) suspended in IDTE Buffer at a concentration of 100 µM. Templates were modified with an amino group on the 3’ end to prevent template extension. All desalted trigger oligonucleotides were purchased from Eurofins Genomics (Louisville, KY) suspended at a concentration of 50 µM in TE Buffer. Triggers were diluted in Nuclease-free water in a separate room to prevent contamination.

Özay B., Robertus C.M., Negri J.L, & McCalla S.E. (2018). First Characterization of a Biphasic, Switch-like DNA Amplification. The Analyst, 143(8), 1820-1828.

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