标签归档：seq

EnGen® Seq1 Cas9 |NEB酶试剂 New England Biolabs

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产品信息

EnGen Seq1 Cas9 from Streptococcus equinus is an RNA-guided DNA endonuclease that catalyzes site-specific cleavage of double-stranded DNA (dsDNA). Targeting requires the formation of a ribonucleoprotein complex of Cas9 and a 116-nucleotide single guide RNA (sgRNA). The sgRNA encodes a 20-nucleotide sequence with complementarity to the DNA target strand immediately upstream of a 5´– NAGA –3´ protospacer adjacent motif (PAM) on the non-target strand. DNA cleavage by EnGen Seq1 Cas9 produces a double-stranded break occurring 3 nucleotides upstream of the PAM. EnGen Seq1 Cas9 has Simian virus 40 (SV40) T antigen nuclear localization signals (NLS) at both the N- and C-termini of the protein.

Figure 1. Schematic representation of S. equinus Cas9 nuclease complexed with a single guide RNA and target DNA

EnGen® Seq1 Cas9 |

S. equinus Cas9 (Seq1 Cas9) protein is shown in beige, single guide RNA (sgRNA) is depicted in blue, and the DNA is shown in grey, green, and red. The DNA target, or protospacer, is shown in green and the base pairing complementarity of the target strand with the guide RNA in the R-loop is illustrated. The protospacer adjacent motif (PAM) which is required for Cas9 binding to DNA is shown in red and the locations of DNA strand cleavage are indicated with black triangles.

产品来源

An E. coli strain that carries the cloned Cas9 gene from Streptococcus equinus with N- and C-terminal Simian virus 40 (SV40) T antigen nuclear localization signal (NLS) and a C-terminal 6XHis tag.

产品类别:
Genome Editing Products,
Programmable Nucleases Products

应用:
Genome Editing Applications

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • M0668T     -20    
        EnGen® Seq1 Cas9 M0668TVIAL -20 1 x 500 pmol 20 µM
        NEBuffer™ r3.1 B6003SVIAL -20 1 x 1.25 ml 10 X

  • 特性和用法

    反应条件

    1X NEBuffer™ r3.1
    Incubate at 37°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)

    贮存溶液

    300 mM NaCl
    10 mM Tris-HCl
    0.1 mM EDTA
    1 mM DTT
    50% Glycerol
    pH 7.4 @ 25°C

    热失活

    65°C for 5 minutes

  • 相关产品

    相关产品

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    • Quick-Load® 紫色 1 Kb Plus DNA Ladder
    • Q5® 热启动超保真 2X 预混液
    • Monarch® PCR & DNA 纯化试剂盒（5 μg）
    • e2050-hiscribe-t7-quick-high-yield-rna-synthesis-kit
    • NEBuilder® 高保真 DNA 组装克隆试剂盒
    • NEBuilder® 高保真 DNA 组装预混液
    • EnGen® 突变检测试剂盒

  • 注意事项

    1. 20 µM 等于 3.22 mg/ml

操作说明、说明书 & 用法

  • 操作说明

    1. In vitro digestion of DNA with EnGen® Seq1 Cas9 (NEB #M0668)

FAQs & 问题解决指南

  • FAQs

    1. Where is the nuclear localization signal on EnGen® Seq1 Cas9 located?
    2. Which nuclear localization signal is fused to EnGen® Seq1 Cas9?
    3. What is the difference between EnGen® Seq1 Cas9, EnGen Sau Cas9 (NEB #M0654), and EnGen Spy Cas9 NLS (NEB #M0646)?
    4. Why do I observe incomplete digestion/editing?
    5. Why does digestion/editing efficiency differ between two different single guide RNAs?
    6. Does NEB provide plasmids for sgRNA cloning?
    7. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?
    8. Can mutations generated with EnGen Seq1 Cas9 be detected using T7 Endonuclease I (NEB #M0302) or the EnGen Mutation Detection Kit (NEB #E3321S)?
    9.  Can sgRNA for use with EnGen Seq1 Cas9 be generated using the EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322)?
    10. How do I design a single guide RNA for use with EnGen Seq1 Cas9?
    11. How do I dilute the enzyme to 1 μM for in vitro reactions?