By Koichi Kawakami, E. Elizabeth Patton, Michael Orger
This moment version info new rising components of zebrafish learn targeting genetics and genomics, recommendations for constructing and studying zebrafish illness versions, and techniques for neuroscience. Zebrafish: equipment and Protocols, moment Edition publications readers via tools for mutagenesis and genome modifying in zebrafish, applications of GFP-expressing transgenic fish, options for melanoma versions, imaging of an infection and host-pathogen interactions, metabolism and delivery of lipids, and the constitution and serve as of neural circuits and their function in producing habit. Written within the hugely profitable Methods in Molecular Biology series structure, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, with no trouble reproducible laboratory protocols, and pointers on troubleshooting and fending off identified pitfalls.
Authoritative and functional Zebrafish: equipment and Protocols, moment Edition is an invaluable supplement to the 1st ebook for brand spanking new and skilled zebrafish researcher alike.
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Additional resources for Zebrafish: Methods and Protocols
Homologyindependent repair mechanisms thereafter lead to the integration of the donor plasmid into the endogenous locus. Indel mutations at the 5′ and 3′ junctions of the genomic target site and the donor Targeted DNA Integration in Zebrafish a 35 b R1 genomic locus genomic locus microinjection F1 DSB - donor plasmid - sgRNA(s) - Cas9 mRNA sgRNA binding sites R2 reporter F2 DSB bait sequence reporter donor plasmid c Homology independent repair forward integrated donor R2 R1 genomic locus reporter F1 F2 5’junction reverse integrated donor F1 5’junction R1 genomic locus reporter F2 3’junction R2 3’junction Fig.
Transcribe the sgRNA using the T7 MEGAscript kit (Life Technologies) as described in the manufacturer’s instructions, and incubate the reaction mixture at 37 °C for 4 h. 2. Add 1 μl TURBO DNase (provided in the kit) to the reaction mixture and incubate at 37 °C for 15 min. 3. To recover the sgRNA, purify the reaction mix using the RNeasy Mini kit according to the manufacturer’s RNA cleanup instructions. Elute two consecutive times with 30 μl each step. Measure the RNA concentration using a spectrophotometer.
Select and pool 10–20 injected and 10–20 un-injected control embryos at 48 h post fertilization. 8. Isolate genomic DNA from the two pools of embryos as described previously [26, 54]. 9. Perform PCR on the target genomic locus in both samples (see Note 18). 42 Thomas O. Auer and Filippo Del Bene 10. Run the PCR products on a 1 % agarose gel to check for the size of the PCR products. 11. Cut and purify the PCR products of the amplified locus using a gel extraction kit, and elute in a final volume of 15 μl.