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S. pyogenes Cas9 NLS

价格 ￥3,400.00 （ 1-2周）
货号 UA079022
规格
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产品介绍

产品介绍 评论(0)

产品规格

  • 表达序列

    APKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK

  • 表达宿主

    E.coli

  • 分子量

    The recombinant SpCas9 NLS consists of 1399 amino acids and has a predicted molecular mass of 161.7 kDa.

  • 纯度

    ≥ 97% by SDS-PAGE gel analyses.

  • 内毒素含量

    <0.1EU/μg

  • 活性

    Measured by its ability to cleave a targeted DNA substrate. Recombinant S. pyogenes Cas9 NLS achieves >90% substrate cleavage, as measured under the described conditions.

  • 标签

    No Tag

  • 性状

    Liquid

  • 缓冲体系

    10 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.1 mM EDTA, 1.0 mM TCEP, 50% glycerol.

  • 储存条件

    Please use a manual defrost freezer and avoid repeated freeze-thaw cycles.

    24 months from date of receipt, -20 to -70 °C as supplied.

    12 months, -20 to -70 °C under sterile conditions after opening.

操作步骤

  • Activity Assay Protocol

    Materials

    Assay Buffer: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 µg/mL BSA, pH 7.9

    Recombinant S. pyogenes Cas9 NLS

    DNA Substrate: PBR322 vector digested with EcoRI Synthetic sgRNA, targeting sequence:, targeting sequence: GAGGCAGACAAGGTATAGGG

    Ethidium Bromide, 10 mg/mL

    Ultrapure DNase/RNase-Free Distilled Water, to prepare Assay Buffer

    Assay

    1. Prepare RNP Complex:

    a. 600 nM sgRNA (6 µL addition from 3 µM stock prepared in Assay Buffer)

    b. 0.25μg rSpCas9 NLS

    c. Add Assay Buffer for a final RNP Complex volume of 26.5 µL

    d. Incubate for 5 minutes at 37 °C.

    2. Mix RNP Complex with 3.5 μL of 8.6 ng/µL of DNA Substrate (diluted in Assay Buffer, if possible).

    3. Incubate for 1 hour at 37 °C.

    4. Incubate for 10 minutes at 65 °C to dissociate enzyme from DNA.

    5. Load total reaction with loading dye on a 1% agarose gel.

    6. Run gel at 140 V for 40 minutes.

    7. Soak gel in 200 mL TAE with 150 μL of 10 mg/mL ethidium bromide for 1 hour.

    8. Use imaging software to detect and quantify hydrolysis of the DNA substrate.

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