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查看更多产品信息 GeneArt™ Platinum™ Cas9 Nuclease (3 µg/µL) - FAQs (B25641)
13 个常见问题解答
利用 GeneArt CRISPR核酸酶载体（https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-crispr/crispr-nuclease-vector.html）引起双链DNA断裂，同时转染基于质粒的供体修复模板。您的供体修复模板质粒将会包含希望引入的序列并在两端具有至少500bp（或更长）的序列，从而实现序列的高效同源重组。
都可以。但为了提高效率，最好使用较长的同源臂（在外源DNA的两端至少500 bp（或更长））。同源长度取决于片段长度且需要测试。ssDNA可能更容易出错或选择NHEJ途径进行修复。针对这种应用，我们提供Invitrogen GeneArtStrings dsDNA片段（1–3 kb）。
We recommend using the Neon Transfection System or the Lipofectamine CRISPRMAX Cas9 Transfection Reagent.
Please see the Lipofectamine CRISPRMAX Reagent manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0014545_lipofectamine_crispermax_QR.pdf) for protocol details or contact techsupport@thermofisher.com.
The Lipofectamine CRISPRMAX Reagent combined with the proprietary enhancement properties of the Lipofectamine Cas9 Plus Reagent leads to efficient complex formation with the Cas9-gRNA ribonucleoprotein, for best delivery to the nucleus, helping to ensure high gene editing frequency for a wide range of cell types.
Although the Lipofectamine CRISPRMAX Reagent was developed for the delivery of the Cas9-gRNA ribonucleoprotein, there is potential for other protein complex applications.
The Lipofectamine CRISPRMAX Reagent was developed to efficiently deliver the Cas9-gRNA ribonucleoprotein complex. We recommend that you first deliver the donor plasmid with Lipofectamine 3000, then follow with delivering the Cas9-gRNA complex with Lipofectamine CRISPRMAX Reagent for best editing efficiency.
For transfecting of the Cas9 protein, we would recommend using the Neon transfection system or Lipofectamine CRISPRMAX Cas9 Transfection Reagent. For transfection of mRNA, we would recommend using Lipofectamine MessengerMAX Transfection Reagent. For transfection of CRISPR vectors, we would recommend using Lipofectamine 3000 Transfection reagent.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Our CRISPR products are optimized for mammalian systems, and have not been optimized for plant systems. However, we know researchers are doing that kind of work. While we have not tested our CRISPR products for plants, our new protein format would be ideal since it does not need to be translated and transcribed in the cell, so no plant-specific promoters required.
The buffer composition for the Platinum Cas9 protein is as follows:
10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.6 mM TCEP, 50% glycerol. Please note that we can make custom formulations if necessary.
The Precision gRNA Synthesis Kit (Cat. No. A29377) includes primers for synthesis of gRNA targeting safe harbor locus HPRT. We also have a fully processed, fully validated HPRT gRNA with GCD primers for confirmation of cleavage available from our custom services team.
Create a double-stranded DNA break using the GeneArt CRISPR Nuclease Vector (https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-crispr/crispr-nuclease-vector.html), while simultaneously transfecting your plasmid-based donor repair template. Your donor repair template plasmid will contain the sequence you wish to introduce that is flanked by at least 500 bp (or more) of sequence, which results in efficient homologous recombination of your sequence.
All of them may work, but for better efficiency, a longer homology arm is better (at least 500 bp (or more) on either side of the exogenous DNA). The homology length is dependent on the size of the fragment and will need to be tested. ssDNA may be error-prone or choose NHEJ. We offer the Invitrogen GeneArt Strings dsDNA fragments (1-3 kb) to assist with this type of application.