...recombination of bacterial plasmid DNA: electron microscopic analysis of in vitro intramolecular recombination. ...One of the most frequent problems that I help troubleshoot involves viral vectors and plasmids with repeating...testing your prep can save you the headache of troubleshooting failed experiments. Pick the right strain While...backbone.   Ready, set, grow! Our 5 Tips for Troubleshooting Viral Transductions will help with the next...

...fluorescent protein reporter under the control of promoters that control cell cycle gene expression.  Additional...genotype the patient and rapidly test the variants in vitro against different drugs and dosages.  Michal Bernach...-up approach will allow me to create a fully controllable and reproducible set up,” Bernach explains. ...containing unstable fluorescent proteins under the control of promoters responsive to water or nutrient levels...

... pH. We compared the dry ice-stored samples to controls that were only stored in a -80°C freezer and not...). Before thawing, all the samples were at the control pH of 7.4 as indicated by color (data not shown...thaw. We compared vented and non-vented tubes to controls that were not exposed to dry ice. Does venting...between 0 and 96 hours. We compared these samples to controls that were only stored at -80°C. We then thawed...period of at least 96 hours had the same pH as control tubes that were never stored on dry ice/exposed...

...same correlation, but it wouldn’t be causal. To control for a number of differences between those papers...methodologies, or qualities of research our adjustment controls for them.   So, for example, in 2010, 10 percent...are lots of other factors that could still be controlled for. As an example, plasmid-related areas of ...see such a big difference in citations after controlling for journal. And so, while our quantitative analysis...research tools, computing performance, and randomized control trials in innovation. Visit his website at: http...

... in the general research community. Phillips Petroleum Company first commercially developed K. phaffii...operates as an obligate aerobe. It’s also a methylotropic yeast, meaning it can utilize methanol as its...peroxisomes are where methanol metabolism occurs in methylotrophic organisms. K. phaffii is an excellent model...and NHEJ is an error-prone pathway frequently introducing frameshift knockout mutations. Thus, if genetic... Susanna Stroik...

...streamline identification. While the Zhang lab controlled their multiplicity of infection so that a single...disease depends on both genetic and expression control systems to mimic illness on a molecular level. ...understand a pathway, or just looking for the best astrocyte differentiation protocol! Ready to try it out...either with or without mCherry and GFP fluorescent controls. Alternatively, you can order individual plasmids...  Susanna Stroik...

...go of the DNA. ABEs are somewhat slow and need a strong Cas9 domain to “hold on” long enough to the DNA...that expresses gene III. Gene III is under the control of a T7 RNA polymerase and cannot be expressed ...the deaminase TadA-7.10 A mutagenesis plasmid controlling the phage mutation rate Phage infection results...editing speed. Created with BioRender.com. Introducing ABE8e - a fast and flexible adenine base editor... activity. To mitigate this drawback, the lab introduced an additional mutation (V106W) into TadA-8e and...

... use amplicons in ITR as we have found this to strongly bias titer. We are also adopting ddPCR, which ...the AAV.PHP.eb for neuron, oligodendrocyte, and astrocyte specificity and would like to get it working in...transfection efficiency when delivering these viruses via retro-orbital vs. tail vein systemic injections? A: We...difference in AAV transduction efficiency between retro-orbital versus tail vein injections.   Q10: Have...labs have tried to transfer PHP.eB's enhanced CNS tropism to other serotypes, but reproducible success remains...

...skills in strong relationship-building and influence. You’ll need a large deal of influence and strong relationship-building...software product management was either. Coming from a strong background of undergraduate research straight to...energized from interacting with others, and have strong communication skills to tailor your message to ... the details, but the developers have it under control. 9:30 AM--Back-to-back meetings. Meet with my boss...

...study the effect of gene knockout in vivo and in vitro, as well as to insert targeted mutations through...separated by 28 base Csy4 recognition sites from a polycistronic transcript. Although this method shortens construct...requires coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may have off-target... of gRNA cleavage.   Xie et. al constructed polycistronic glycine tRNA-gRNA genes (PTGs) in the forms ...Bacteria and viruses have illustrated the power of polycistronic gene organization, and its translation to CRISPR...

...after the plasmids have been introduced by transfection or electroporation. All you do to create a protein...type of genome editing tool with which one can introduce changes into the genomes of mammalian or other... create a pair of designer proteins that will introduce a double-strand break in the genome anywhere you...double-strand breaks, there is a possibility of introducing a mutation somewhere else in the genome. It remains...

...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...

...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...

...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...

...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....

...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...