We narrowed to 717 results for: CaS;

...Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. Enzymatic assembly of DNA molecules...modularity to overlap-directed methods (Casini 2014). In this case linker sequences are added before the...In this case, rather than using a restriction enzyme or PCR to make linearized vector, the Cas9 enzyme ...standard sequences with overlap regions, such as in the case of MODAL (modular overlap-directed assembly with...restriction site for linearization. This is a perfect case for the use of Gibson assembly combined with the...assembled into a vector using Gibson assembly. In this case the vector was PCR amplified to contain regions ...Li K, Wang B, Jin S, Reiser M, Lockey RF. CRISPR/Cas9 nuclease cleavage combined with Gibson assembly ...

...A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast 1 and then selected with...Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with lentiCas9-Blast... express Cas9 and are included as a control (WT). Vinculin is a loading control. 1 lentiCas9-Blast was...additional 3 weeks under blasticidin selection. Anti-Cas9 Western blotting was performed on whole-cell extracts...

...Biotechnology.       References Degeling MH, Maguire CA, Bovenberg MSS, Tannous BA (2012) Sensitive Assay...to discard the infected cell culture flasks. In cases where the culture is irreplaceable or very expensive...

...carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering." Cell 154 (2013):1370... 47327 pET-28b-Cas9-His 6xHis (C terminal); NLS (C terminal) 62933 pET-Cas9-NLS-6xHis 6xHis.... recommend ribonucleoprotein (RNP) complexes of Cas9 protein and the separated guide RNA components (..., we’ll use the example of inserting a 2A-FlpO cassette into the Fgf8 gene. Miura et al. (2018) suggest...insertion site to maximize HDR efficiency. In this case, we’re fortunate that there’s a guide that directs... and cleavage sites (slashes) are shown. In this case, the green guide would be selected, since it cleaves...increase T7 promoter activity. To avoid re-cutting by Cas9 after editing, make sure that the repair template...

...consisting of: 5.0 g yeast extract 10.0 g peptone from casein 10.0 g sodium chloride 12.0 g agar-agar 1 L Sterile...actually make a bit more than 200 mL (~220 mL) just in case we spill anything or have small errors in measurement...the autoclave. Transfer the sterile water (in our case 220 mL) to the same bottle and swirl to form a medium...s manufacturer to make sure this is true in your case. Retrieve your molten agar mix from the autoclave...positive and negative test results. Sample Data In all cases below (-) indicates that the tested strain is not...

...protein concentration in the lysate, running a precast SDS-PAGE gel, and immunoblotting....protein concentration in the lysate, running a precast SDS-PAGE gel, and immunoblotting. Sharing speeds...23227 β-mercaptoethanol 4X protein loading buffer Precast SDS-PAGE gel SDS-PAGE running buffer Prestained...min at 100 °C . Section 3: SDS-PAGE Prepare the precast gel as follows: Remove the gel from the plastic...

...in Vibrio cholerae that encodes a Type I-F CRISPR-Cas system. This system has four major components: 1)...crRNA), 2) four proteins (TniQ, Cas8, Cas7, Cas6) that forms the QCascade DNA-targeting module with the ... transposons encode reporter genes, mutagenic cassettes, or barcodes. When transposons are delivered to...For the INTEGRATE system to transpose, first the QCascade complex surveys the genome and binds its target...molecule, and inserts it ~50 bp downstream of QCascade’s genomic target site (Fig. 3B). The INTEGRATE ...of RNA-guided transposition with INTEGRATE. The QCascade complex surveys the genome and binds its target...molecule, and inserts it ~50 bp downstream of QCascade’s genomic target site.  T-RL= transposon right-...

...allow positively charged ions such as Na+, K+, and Ca++ to passively enter the cell with the concentration...function through multiple intra-cellular signaling cascades. Optical methods for controlling GPCR activation...control GPCR activation and downstream signaling cascades (reviewed in Abreu & Levitz, 2020), including ...extracellular domain which activates the native signaling cascade of the intracellular part of the receptor, similar... out important branch points along a signaling cascade that may go awry in a disease state.      ...

...and shift its activity back to polymerase. In our case, dGTP will be used in the reaction, meaning that...there are no gaps in the final assembly. In this case, 15 bp of homologous sequence is used, plus a minimum...following the manufacturer's instructions. In this case, we will include the free nucleotide dGTP in the...

...seeded the day before transduction. Note that in that case, polybrene should only be added at the time of viral...sensitive to lentivirus or to polybrene, in which case this incubation step can be reduced to 8 h and the...serial dilutions of the lentiviral vector pHAGE EF1α dCas9-KRAB . The following day, cells were treated with...

...hM1Dq hM5Dq Human M3 muscarinic G αq CNO* Increase Ca 2+ Neuronal burst firing Armbruster et al., 2007 ...

...A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast and then selected with...formed by expansion of single cells for 13 days. lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid...

...Find out more at www.projbridge.com. Walnut Creek, CA, USA 37.9101°N 122.0652°W – Steph Hays When I marched...22 to join in the March for Science.  Cold and overcast with intermittent rains, it was weather befitting...

...Addgene. 10. Port, Fillip, et al. “Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering...humanized Cas9, D10A nickase mutant (Cas9n), dCas9 (transcriptional repression), and Fok1-dCas9 (dimeric...options include humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator...including Standard PolIII-gRNA cassette, Csy4-cleavable cassette, and PTG cassette.    Find gRNA multiplexing... then digested using BsmBI and ligated into Cas9 or dCas9-containing destination vectors. Destination ... encoding the gRNAs and Cas9. As with the Gersbach lab plasmids, multiple Cas9 variants are available:...the multiple gRNAs and Cas9 together into a single plasmid. Although only wt hCas9 entry vectors are supplied...

... how to perform gel electrophoresis. Equipment Casting tray Well combs Voltage source Gel box UV light...prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.). Caution HOT! Be ...

...of a hairpin structure within your primer. In our case, we will add TAAGCA, resulting in a final Forward...to the sequence to be amplified (the ORF in this case), not the Tm of the entire primer. If you are amplifying...

...antibody concentration may have been too low. In this case, use higher antibody concentrations. If the reaction...antibody concentration may have been too high. In this case, use lower antibody concentrations. The ideal range...

...Root Cas9 Lentivirus Cas9 viruses can be used to interrogate the genome in a variety of ways. Cas9 proteins... 52962 lentiCas9-Blast Sp Cas9 Cut Blasticidin Expresses human codon-optimized S. pyogenes Cas9 protein...backbone. Zhang Cas9 and Accessories for Activating Gene Expression Catalytically-dead Cas9 (dCas9) can be fused...Resistance Activity PI 61422 dCAS9-VP64_GFP dCAS9 (D10A, H840A) none Expresses dCAS9-VP64 activator with 2A ...lenti dCAS-VP64_Blast dCAS9 (D10A, N863A) Blasticidin 3rd generation lenti vector encoding dCAS9-VP64 ...-targeting controls. This backbone contains SpCas9. SpCas9 and 76,441 unique sgRNAs targeting 19,114 human...plasmids resource page . Cas9 for Editing Genomic Sequences To perform genome edits, Cas9 nuclease is used to...

...Anti-CASPR/Neurexin IV [K65A/2R] CASPR/Neurexin IV Mouse IgG2a 206606 Anti-CASPR2 [K67/11R] CASPR2 Human...190491 CASK scFv [K56A/50] K56A/50 scFv CASK Human Mouse 190492 CASK scFv [K56A/57] K56A/57 scFv CASK Human...177445 Anti-CASPR/Neurexin IV [K65/35R] CASPR/Neurexin IV Rat Mouse IgG2a 177446 Anti-CASPR2 [K67/25R] ...219448 Anti-CASPR/Neurexin IV [K65A/30R] CASPR/Neurexin IV Rat Mouse IgG2a 219449 Anti-CASPR/Neurexin IV...SAP97 Rat Mouse IgG2a 128625 Anti-CASPR/Neurexin IV [K66/38R] CASPR/Neurexin IV Rat Mouse IgG2a 128626...Pan-KChIP K+ channel Mouse IgG2a 177443 Anti-CASK [K56A/50R] CASK Human Mouse IgG2a 177444 Anti-KChIP2b K+...Synaptotagmin-6 Mouse Mouse IgG2a 188217 Anti-CASK [K56A/57R] CASK Human Mouse IgG2a 188218 Anti-Kv2.1 K+ channel...

... for shuttling U6-guide cassettes to make dual guide expressing plasmids dCas9-KRAB-MeCP2 fusion for gene...enhancer sequence repression dCas9-VPR fusion for gene activation CRISPR/Cas9 Plasmid Variants Figure 1:...175574 express dCas9-KRAB-MeCP2 for repression. Plasmids 175571 and 175572 express dCas9-VPR for activation...in retinal development the lab utilizes a CRISPR/Cas9 based approach to knockout, repress, and activate...taken advantage of the Zhang lab all-in-one (guide/Cas9/reporter) pX458 plasmid family. They have made several...plasmids to insert a second guide within a U6-guide cassette. This process is compatible with all pX458 family...Sources Original pX458 backbone ( Addgene #48138 ) dCas9 + KRAB-MeCP2 ( Addgene #110821 ) VPR ( Addgene #63798...