pMD19T-vector kit(TAKARA, 3271) Equipment Centrifuge(RT and 4℃) Vortex OneDrop OD-1000+ Spectrophotometer Thermocycler Thermomixer Thermo-controlledwater bath(37℃,42℃ and 58℃) Procedure Constructionof sgRNA expression vectors 1. Design of...
pMD19T-vector kit(TAKARA, 3271) Equipment Centrifuge(RT and 4℃) Vortex OneDrop OD-1000+ Spectrophotometer Thermocycler Thermomixer Thermo-controlledwater bath(37℃,42℃ and 58℃) Procedure Constructionof sgRNA expression vectors 1. Design of paired sgRNA oligos. Select paired sgRNAs in a tail-to...
lenti viral vector harboring one cas9 protein and two guide RNA expressing cassettes is generated. In this protocol, three plasmids are involved. lentiCRISPR-Dual-puro and lentiCRISPR-Dual-GFP contain cas9 and one Guide RNA expressing ca...
3 Construction of the plant binary expression vector pCAMBIA1300-pYAO-Cas9-NtREV Hyg 基因特异引物对 Hyg‑F 和 Hyg‑R 进行 PCR 扩增 检测(表 1),结果(图 4)显示,48 株待检测材料 中有 44 株材料的 DNA 模板扩增出约 700 bp 目的条 带,表明成功获得 44 株 REV 转基因编辑植株,总 体转化效率...
Vector construction Detailed descriptions of the vector construction are provided in Additional file2: Methods S1. All primers used in this report are listed in Additional file1: Table S1. Golden gate method to construct a vector expressing one or two gRNAs ...
Finally, two sgRNA expression cassettes were ligated into pYLCRISPR/Cas9Pubi-H vector via Golden Gate ligation method [40]. Oligonucleotide primers used for recombinant pYLCRISPR/Cas9 vector construction are listed in Additional file 6: Table S3. Plant Transformation The confirmed pYLCRISPR/Cas9Pubi...
U3 and U6 promoters from rice (Os) and Arabidopsis(At) and the sgRNA sequence are separated by the vector backbone, to avoid amplification of the uncut plasmids by PCR with short extension time during the construction of sgRNA expression cassettes. Cutting(small arrows) with Bsa I produces ...
The preferred miR-126 gene of the invention downstream of the CRISPR-Cas9 target sequence, was designed and synthesized for SgRNA single strand of the target sequence, and constructs the vector by transfected 293T cell line, SgRNA with trRNA constituting specific recognition structure, such that ...
In this study, we sought to simplify CRISPR/Cas9 mediated gene KO to obviate the need for vector construction. Our motivation was to develop a simple, cost-effective, efficient and scalable gene KO approach that parallels the simplicity of siRNA transfection. We showed that the co-transfection ...
In this study, we sought to simplify CRISPR/Cas9 mediated gene KO to obviate the need for vector construction. Our motivation was to develop a simple, cost-effective, efficient and scalable gene KO approach that parallels the simplicity of siRNA transfection. We showed that the co-transfection ...