bbs1 digested pspcas9 bb 2a puro px459 v2 0  (Addgene inc)

Journal: Genes

Article Title: Heterozygous Knockout of ARID4B Using CRISPR/Cas9 Attenuates Some Aggressive Phenotypes in a Breast Cancer Cell Line.

doi: 10.3390/genes14122184

Figure Lengend Snippet: Figure 2. Construction and validation of the CRISPR/Cas9 system. (A) The structure of the ARID4B protein with a total of 1312 amino acids and the site where the mutations were generated in position 939, indicated as a hotspot. (B) Design of the sgRNA and validation of the ligation in the pX459 vector by Sanger sequencing. Only underlined region is shown. Red for T, Black for G, Blue for C, and Green for A. (C) Confirmation of the presence of genetic editions in the pool of MCF-7 cells after transfection. The surveyor assay produces a cut in the PCR product, generating two fragments indicated by arrows. (D) Validation of the presence of genetic editions in isolated clones. Here, 3, 10, and 21 represent the clones positive for mutations in ARID4B.

Article Snippet: Both oligos were aligned and cloned in Bbs1 digested pX459 (62988, Addgene, Cambridge, MA, USA).

Techniques: Biomarker Discovery, CRISPR, Generated, Ligation, Plasmid Preparation, Sequencing, Transfection, Isolation, Clone Assay