...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...

...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...

...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.      ...

...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-...

...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...

...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...

...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...

...light that your substance maximally absorbs. In the case of nucleic acid (DNA and RNA), the maximal absorbance...

... deletions in mammalian cells lines using CRISPR/Cas9 Genomic Deletions with CRISPR Protocol Over-Agar...

... not dislodge any pellets or re-mix liquids. In cases where you are separating solids from a liquid, pellets...

...transduced with a range of dilutions of pHAGE-TO-dCas9-3XmCherry . 72 h post transduction, cells were assayed...

...per 10 cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this would...

... or can come preloaded into tip boxes. In either case, once assembled into their boxes, pipette tips can...

...pAC91-pmax-dCas9VP64 : dCas9VP64 on pmax expression vector. 48224 pAC92-pmax-dCas9VP96 : dCas9VP96 on pmax...-pCR8-dCas9VP48 : dCas9VP48 on Gateway donor vector pCR8/GW/TOPO. 48218 pAC84-pCR8-dCas9 : dCas9 on Gateway... pAC93-pmax-dCas9VP160 : dCas9VP160 on pmax expression vector. 48226 pAC94-pmax-dCas9VP160-2A-puro : dCas9VP160...pAC147-pCR8-dCas9VP64 : dCas9VP64 on Gateway donor vector pCR8/GW/TOPO. 48220 pAC148-pCR8-dCas9VP96 : dCas9VP96...components, the dCas9 activator protein and the single guide RNA (sgRNA). While the dCas9 protein provides... VP160 with 10. dCas9 activators and sgRNAs can be separately transfected (pmax dCas9 activators [48223...below: Table 1. dCas9 Activators sgRNA Dual Expression ID Plasmid 48236 pAC2-dual-dCas9VP48-sgExpression ...