...you multiplex - we’ll use mammalian plasmids to introduce you to some of your potential options and cloning...transcript You can also multiplex gRNAs via a polycistronic transcript. Rather than being transcribed from...option described below, plasmids for making polycistronic gRNAs are also available from the Yang lab for...cannot be released, adding temporal and/or spatial control to the system. pSQT1313 from the Joung lab allows...Plasmids: These plasmids are similar to the Csy4 polycistronic system described above, except that they use...gRNAs are flanked by glycine tRNAs to create polycistronic glycine tRNA-gRNA (PTG) constructs. Eukaryotic...

...quantity and quality of your plasmid prep is by spectrophotometry. Achieving the highest-quality DNA requires...plasmid copy number   Pro tip! If you are having trouble getting sufficient plasmid DNA, you may need to...factor label Non-silica based, avoids use of chaotropic salts   What are all these other kit types...manufacturers provide specs, protocols, and quality control information, but the level of detail can vary. ...the rationale behind each step and provide troubleshooting advice. In these cases, it can be helpful to... instructions and extensive references for troubleshooting. If you need a high level of consistency between...

...autophagy. Morphological assays Electron microscopy: Electron microscopy is a traditional method for...this is indicative of protein degradation). To control for protein degradation due to other pathways, ...at various stages of degradation. Additionally, electron microscopy analysis is time consuming. GFP-tagged...emit red and green fluorescence. After 4 hours of nitrogen starvation to induce autophagy, red but not green...

...native regulatory elements. Controls for RaPID experiments RaPID negative controls include both scrambled ...occurring complexes present in the cell, it can also introduce non-physiological RNA-protein interactions due....  In as little was one minute BASU generated a strong streptavidin signal (~250-fold enrichment) when...when compared to a scrambled RNA motif control). BioID2 had no detectable signal at the same time point....

...fluorescence microscopy, flow cytometry, or spectrophotometry. The range of fluorophores available for antibody...capable of producing a visible signal, but it has a strong affinity for avidin and streptavidin, both of which...modifying the antibody sequence or that rely on strong protein-protein interactions with other peptides...of non-canonical amino acids or peptide tags. 5) Strong protein-protein interactions with other peptides...of antibody conjugation. But hopefully, this introduction provides a good base from which to dive deeper...

...low-throughput methods such as patch-clamp electrophysiology, a technique that’s used to measure electric...selected for characterization with patch clamp electrophysiology. All thirty were functional and their measured...experiments, neurons expressing ChRgers produced stronger photocurrents at low light intensity than existing...the mice to increase their nose-poking behavior. Strong nose-poking behavior is exactly what was seen with...self-stimulation response. This result showed that ChRger2 stronger photocurrent properties make it better suited ...

...If you know the sequence of a virus, you could introduce some of its dsRNA intermediates into a healthy...the inflammatory response associated with the introduction of dsRNA. Synthetic dsRNA Researchers have...time, even before we knew about all the diverse in vitro production mechanisms. siRNA, also known as RNAi...and repetitive regions, such as telomeres and centromeres. These genomic regions are prone to persistent...  Susanna Stroik...

...biology - even a piece of wood - there’s a lot of control spatially as to what’s happening at different points...cellulose fibers, pattern, shape. All of that is controlled by regulatory networks in the plant. If we want... hundreds of genes and regulation and resource control - everything that’s necessary on that scale.  If... terminators. That gives us enough hardware to control hundreds of genes. We’ve created enough regulatory...transcriptional and translational activity a cell has and control how much it taxes the resources of a cell. The ...

...reproduction. Both physiological processes are controlled by deliberate and compulsory actions instigated...corticosteroids, progesterone, androgens, and estrogens), fat-soluble vitamins A and D, thyroid hormone...complex can serve as a rate-limiting conduit to control the actions of the whole complex.  For example,...adipogenesis and macrophage activation. Our mass spectrometry-affinity purification proteomic profiling of...N. J.and O'Malley, B. W. (2002). Combinatorial control of gene expression by nuclear receptors and coregulators...

...derived from the E. coli Ec48 retron RT and the S. pombe Tf1 retrotransposon RT, respectively. Their small...less than 10%. In contrast, the original paper introducing prime editing demonstrated ~20-50% efficiency...fold (Anzalone et al., 2019). PE3, as originally introduced, relies on the PE2 enzyme. PE4 and PE5: Engineering...ligases to act first. PE4 and PE5, as originally introduced, rely on the PE2 enzyme. PEmax: Optimizing prime...found that the mutations that emerged depended strongly on the target edit (Doman et al., 2023). Therefore...