Journal: Current protocols in molecular biology

Article Title: CRISPR/Cas9-Directed Gene-Editing for the generation of loss-of–function mutants in high-throughput zebrafish F O screens

doi: 10.1002/cpmb.42

Figure Lengend Snippet: DR274 sgRNA digestion and cloning scheme. A. The DR274 sgRNA plasmid has two BsaI restriction enzyme recognition sites that cut outside the binding site to leave 4bp overhangs. Complementary oligos are ordered, annealed, and then ligated into the overhang sites. B. Optimal CRISPR target site (Red) in exon 2 of flh gene showing PAM sequence (blue) and cut site (green). Design of oligos for cloning flh target site into the BsaI digested DR274 backbone.

Article Snippet: Cat# 74104 100% RNase Free Ethanol Obtain DR274 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 Order constructs from Addgene 2 Use an inoculation loop to streak from the Addgene bacterial stab onto LB plates with 50 μg/ml kanamycin.

Techniques: Cloning, Plasmid Preparation, Binding Assay, CRISPR, Sequencing

Journal: Current protocols in molecular biology

Article Title: CRISPR/Cas9-Directed Gene-Editing for the generation of loss-of–function mutants in high-throughput zebrafish F O screens

doi: 10.1002/cpmb.42

Figure Lengend Snippet: Example of sgRNA RNA production as compared DraI digested and uncut plasmid customized DR274 plasmid. sgRNA is approximately 103bp but will not run according to size due to its single stranded nature.

Article Snippet: Cat# 74104 100% RNase Free Ethanol Obtain DR274 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 Order constructs from Addgene 2 Use an inoculation loop to streak from the Addgene bacterial stab onto LB plates with 50 μg/ml kanamycin.

Techniques: Plasmid Preparation