1 / 3

eci2 knockout in Zebrafish using CRISPR-Cas9 System

131028 Mie Meeting Research, Yasuhito Shimada. eci2 knockout in Zebrafish using CRISPR-Cas9 System . The diverse potential application of CRISPR-Cas9 system. Nat Methods. 10: 957-963, 2013 (Figure 2C). Purpose: To construct eci2 knockout zebrafish. The CRISPR Design Tools

cindy
Download Presentation

eci2 knockout in Zebrafish using CRISPR-Cas9 System

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 131028 Mie Meeting Research, Yasuhito Shimada eci2 knockout in Zebrafish using CRISPR-Cas9 System The diverse potential application of CRISPR-Cas9 system. Nat Methods. 10: 957-963, 2013 (Figure 2C) Purpose: To construct eci2 knockout zebrafish. The CRISPR Design Tools ZiFitTargeter Version 4.2 (ZINC Finger Consortium, Joung Lab, Massachusetts General Hospital): Nat Biotechnol. 31: 227-229, 2013 CRISPR Design (Zhang Lab, Broad Institute of MIT and Harvard): Nat Biotechnol. 31: 827-832, 2013 ① ② ③ eci2 mRNA sequence (not genome sequence)

  2. ④ Results Check by Genome-blast Remove the oligo covered to exon-intron boundary ⑤ Positive controls fh: Zebrafish-gRNA-0004 (site #2) gsk3b: Zebrafish-gRNA-0005 (Nat Biotechnol. 2013, Supplementary) Plasmid Purchase from Addgene Cut: Cas9 nucleases Nick: Cas9 nickases Interfere: CRISPR interference (CRISPRi) Active: dCas9-activator fusion

  3. ⑥ Amp Kan Plasmid Construction DR274 linearization using BsaI ⑦ ⑧ 2.1kbp 37℃, 1h 65℃, 20min Electrophoresis on 1.5% Agarose gel Gel Extraction using Wizard SV Gel and PCR Clean-Up System (Promega) Measure the concentration using NanoDrop (35ng/μl in 50 μl) Annealing for insert oligonucleotides using 1 ×annealing solution (Life Technologies) ⑨ Ligation using Ligation-convenience Kit (Wako Pure chemicals) and Transformation Plasmid : Insert DNA = 1 : 5 The size of plasmid = 2147 - extra bp = 2100 bp The MW = 660 x 2100 = 1386000 34.37 ng/ul = 24.8 nM Transformation DR274 (positive control) DR274-linealized Ligated DR274 (gsk3b) 10 min at 16℃.

More Related