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- 2025年10月08日
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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
1年
- 英文名：
EnGen® Spy Cas9 Nickase
- 库存：
999
- 供应商：
上海淳麦
- 规格：
1000U/10000U
EnGen® Spy Cas9 Nickase is a variant of Cas9 nuclease differing by a point mutation (D10A) in the RuvC nuclease domain, which enables it to nick, but not cleave, DNA (1,2). EnGen Spy Cas9 Nickase, like EnGen Cas9 NLS (NEB #M0652), targets DNA using a guide RNA complementary to a site with a 3’ NGG protospacer adjacent motif (PAM). Nicking occurs on the DNA strand opposite the PAM. Double-stranded DNA breaks can be generated with reduced off-target cleavage by targeting two sites in close proximity (generally 0-20 bp apart) and with PAMs facing outward to leave 5’ overhangs. Nicking by EnGen Spy Cas9 Nickase occurs on the DNA strand opposite the target sequence. EnGen Spy Cas9 Nickase contains SV40 T antigen nuclear localization (NLS) sequences on the N and C- termini.
Product Source
EnGen® Spy Cas9 Nickase is expressed as a N-terminal 6XHis-tagged fusion in E. coli.
Reagents Supplied
The following reagents are supplied with this product:
Description
EnGen® Spy Cas9 Nickase is a variant of Cas9 nuclease differing by a point mutation (D10A) in the RuvC nuclease domain, which enables it to nick, but not cleave, DNA (1,2). EnGen Spy Cas9 Nickase, like EnGen Cas9 NLS (NEB #M0652), targets DNA using a guide RNA complementary to a site with a 3’ NGG protospacer adjacent motif (PAM). Nicking occurs on the DNA strand opposite the PAM. Double-stranded DNA breaks can be generated with reduced off-target cleavage by targeting two sites in close proximity (generally 0-20 bp apart) and with PAMs facing outward to leave 5’ overhangs. Nicking by EnGen Spy Cas9 Nickase occurs on the DNA strand opposite the target sequence. EnGen Spy Cas9 Nickase contains SV40 T antigen nuclear localization (NLS) sequences on the N and C- termini.
Product Source
EnGen® Spy Cas9 Nickase is expressed as a N-terminal 6XHis-tagged fusion in E. coli.
Reagents Supplied
The following reagents are supplied with this product:
Advantages and Features
Applications
Paired, offset cleavage of genomic loci
Target-specific DNA nicking
Properties and Usage
Reaction Conditions
1X NEBuffer 3.1
Incubate at 37°C
1X NEBuffer™ 3.1:
100 mM NaCl
50 mM Tris-HCl
10 mM MgCl2
100 μg/ml BSA
pH 7.9 @ 25°C
Storage Temperature
-20°C
Storage Conditions
300 mM NaCl
10 mM Tris-HCl
0.1 mM EDTA
1 mM DTT
50% Glycerol
pH 7.4 @ 25°C
Notes
- 1. 20 µM is equal to ~3.2 mg/ml EnGen® Cas9 Nickase NLS, S. pyogenes. 1 µM is equal to ~0.16 mg/ml.
- 2. This product is compatible with Diluent Buffer B: 300 mM NaCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, 500 µg/ml BSA and 50% glycerol. (pH 7.4 @ 25°C).
References
- Mali, P., et. al. (2013). NatBiotech. 31 (9), 838-8.
- Ran, FA., et. al. (2013). Cell. 154 (6), 1380-9.
上海淳麦生物科技有限公司专注于感受态细胞、质粒、载体、农杆菌、酵母等生命科学领域的产品研发和技术服务支持。
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文献和实验DNA extraction from Mutation Detection Enhancement (MDE) Gel Stained with Silver Nitrate
extension cycle at 72 ˚C for 5 min. PCR reactions were performed in Gene Amp® PCR System 480 (Perkin-Elmer, Norwalk, CT, USA). DNA samples of normal subjects (blood donors) were included as well as negative control (with no DNA sample) in each reaction.
The ribonuclease protection assay (RPA)
, 5X transcription buffer, and RPA template set to RT. For each probe synthesis, add the following in order to a 1.5 ml Eppendorf tube: 1 µl RNasin® 1 µl GACU pool 2 µl DTT 4 µl 5X transcription buffer 1 µl RPA Template Set 10 µl [a-32
ORNL MICROARRAY HYBRIDIZATION PROTOCOLS
Direct labeling of total RNA with Cy3 and Cy5: A. MATERIALS RNeasy® Mini Kit (Qiagen; Cat # 74106) SuperScript II RT (200U/µL) (Life Technologies; Cat # 18064-014) QIAquick PCR Purification Kit (Qiagen; Cat # 28106)
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