Journal: Journal of visualized experiments : JoVE

Article Title: Screening Sperm for Rapid Isolation of Germline Edits in Zebrafish

doi: 10.3791/64686

Figure Lengend Snippet: Table of Materials

Article Snippet: Phosphorothioate bond on the donor at the first three phosphate bonds on both the 5’ and 3’ ends (5’-CCTCTCTCCCTTTCAGAAGCTTCTGCTCATCCGCTGCTTCTGCCTGGACCGAGTGTACCGTGCCGTCAGTGATTACGTCACGC-3’) dnah10 forward primer Sigma-Aldrich DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry (5’-CATGGAACTCTTTCCTAATGAGTTTGGC-3’) dnah10 reverse primer Sigma-Aldrich DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry (‘5-AGTAGAGATCACACATCAACAGAATACAGC-3’) dnah10 synthetic sgRNA Synthego Synthetic sgRNA, target sequence: 5’-GCTCATCCGCTGCTTCAGGC-3’ Electrophoresis power supply Thermo Scientific 105ECA-115 Filter forceps Millipore XX6200006P Fish (system) water Generic n/a Gel electrophoresis system (including casting frame, comb, and electrophoresis chamber) Thermo Scientific B2 Gel imaging light box Azure Biosystems AZI200–01 Gel stain, 10000X Invitrogen S33102 Glass bowl, 250 mL Generic n/a Isolation tanks, 0.8 L Aquaneering ZT080 Microcap capillary tube with bulb, 20 μL Drummond 1–000–0020/CA Minicentrifuge Bio-Rad 12011919EDU Micropipettes, various with appropriate tips Generic n/a Microwave Generic n/a Nuclease free water Promega P119-C Paper towels Generic n/a PCR tubes, 0.2 mL Bioexpress T-3196–1 Plastic spoon, with drilled holes/slots Generic n/a KCl solution, 0.2 M RNAse Free Sigma-Aldrich P9333 p2ry12 forward primer Sigma-Aldrich DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry (5’-CCCAAATGTAATCCTGACCAGT-3’) p2ry12 reverse primer Sigma-Aldrich DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry (5’-CCAGGAACACATTAACCTGGAT-3’) p2ry12 synthetic sgRNA Synthego Synthetic sgRNA, target sequence: 5’-GGCCGCACGAGGTCTCCGCG-3’ Restriction Enzyme 10X Buffer NEB B6003SVIAL NaOH solution, 50 mM Thermo Scientific S318; 424330010 Sponge, 1-inch × 1-inch cut with small oval divot Generic n/a Stereomicroscope Zeiss Stemi 508 Taq polymerase master mix, 2X Promega M7122 TBE Buffer Concentrate, 10X VWR E442 Thermal Cycler Bio-Rad 1861096 Tissue paper Fisher Scientific 06–666 Tricaine-methanesulfonate solution (Syncaine, MS-222), 0.016% in fish water (pH 7.0±0.2) Syndel 200–266 Tris Base, 1M (Buffered with HCl to ph 8.0) Promega H5131 Open in a separate window Table of Materials.

Techniques: Construct, Purification, Sequencing, Electrophoresis, Nucleic Acid Electrophoresis, Imaging, Staining, Isolation