Download e-book for iPad: Engineered Zinc Finger Proteins: Methods and Protocols by Mital S. Bhakta, David J. Segal (auth.), Joel P. Mackay,

Download e-book for iPad: Engineered Zinc Finger Proteins: Methods and Protocols by Mital S. Bhakta, David J. Segal (auth.), Joel P. Mackay,

By Mital S. Bhakta, David J. Segal (auth.), Joel P. Mackay, David J. Segal (eds.)

Among the numerous kinds of DNA binding domain names, C2H2 zinc finger proteins (ZFPs) have confirmed to be the main malleable for growing customized DNA-binding proteins. In Engineered Zinc Finger Proteins: equipment and Protocols, professional researchers from probably the most energetic laboratories during this box current certain tools, suggestions, and views. the amount includes sections protecting the engineering of ZFPs, equipment for the production, assessment, and supply of man-made transcription components (ATFs), tools for the production and overview of zinc finger nucleases (ZFNs), and a suite of different purposes and assays past ATFs and ZFNs, together with zinc finger transposases and ChIP-seq method among different matters. Written within the hugely winning equipment in Molecular Biology™ sequence structure, chapters contain introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and notes on troubleshooting and averting recognized pitfalls. complete and state-of-the-art, Engineered Zinc Finger Proteins: equipment and Protocols goals to help either pro practitioners and new investigators with its important tools and insights as they search to create the subsequent new release of engineered ZFPs and applications.

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Extra info for Engineered Zinc Finger Proteins: Methods and Protocols

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Since pKJ1712 is a low copy number plasmid, 10 mL overnight cultures in liquid LB/kanamycin (30 μg/mL) will yield an adequate DNA concentration when using the QIAgen’s QIAprep Spin Miniprep Kit. 1× EB (a 1× EB stock is provided in the QIAgen kit). 9. To verify uptake of the annealed oligonucleotides in the pKJ1712 plasmid, we digest 5 μL of each candidate DNA with EcoRI and HindIII. As a control, we also digest plasmid pKJ1712 with EcoRI and HindIII. Recombinants that have taken up the annealed oligonucleotide insert should yield five bands of sizes 6,109, 1,006, 963, 431, and Engineering Single Cys2His2 Zinc Finger Domains 39 190 bp.

Check for correct amplification by analyzing 10 μL of the reaction using agarose gel electrophoresis. Purify the amplicon using a PCR purification kit. 3. Digest both the amplicon and pSSA-1-3 using BglII and EcoRI (see Note 7). 4. Ligate the amplicon into the pSSA vector using T4 DNA ligase. 5. Because the duplicated region also makes sequencing of the final plasmid difficult, the junction of the repeated region (containing the ZFN target site) should be first amplified in the following 50 μL PCR reaction: a.

106, 5053–5058. 20. J. (2008) Critical parameters for genome editing using zinc finger nucleases. Mini Rev Med Chem. 8, 669–676. 21. Sera, T. (2009) Zinc-finger-based artificial transcription factors and their applications. Adv Drug Deliv Rev. 61, 513–526. 22. , and Carroll, D. (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics. 161, 1169–1175. 23. R. (2006) Gene transfer of an engineered transcription factor promoting expression of VEGF-A protects against experimental diabetic neuropathy.

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