We narrowed to 1,015 results for: NTS

...literature. Data reported from flow cytometry experiments can be a little challenging to understand if ... and output is generated through measurement of events. An event is something that passes through the ...axis to generate a dot plot (Figure 1). Clustered events are called a population.                           ...left, it might make more sense once you realize events are plotted on an invisible Cartesian plane (right...real-world data is far messier than biorender plots, quadrants can still help me as a reader if I'm struggling...strategy from Barlow-Anacker et al., 2017, with quadrants to help visualize the different populations. Used...populations; this indicates the percentage of total events contained in the gated population. Heat maps are...

... protocol or reagents, do you understand all of the steps? Do you know how the reagents have been validated...preparation) A history of successful funding (fellowship grants) A project with sufficient preliminary data to...couple of months and requires that all of the experiments have been completed. Therefore, to be ready to...postdoctoral mentor. A good mentor typically writes the grants that fund your research, guides the research directions...provide expertise in designing and interpreting experiments, as well as career guidance. In my own experience...the methodology and can have confidence in the reagents and outcomes. This is preferable to finding out...work for your specific assay. "Quick and dirty" experiments often eat far more of your time as they can result...

...fluorescence imaging strategies use widely varying amounts of excitation power (Shaner et al, 2008). In confocal...to predict how fluorophores perform in ‘real experiments’, it is advisable to perform time-lapse imaging...with low excitation power. By repeating these measurements for different fluorescent proteins and by comparing...protein distribution. Therefore, photostability measurements can be performed with soluble fluorescent proteins.... It should be noted that the photostability measurements reported in the literature are performed in ... the experimental designs used to make these measurements and hope this information can help guide your...evolves over time. The second issue is that, in experiments that measure photostability, high excitation ...

...overhangs can be used to assemble multiple fragments - up to 10 fragments are commonly assembled in a single ...increased number of fragments, or the ligation of very small/very large fragments, these problems can ...homology at the ends of DNA fragments to specify assembly order, so fragments with 5’ or 3’ sequence homology...they can be customized to direct assembly of DNA fragments. When designed correctly, the recognition sites... These overhangs specify the desired order of fragments, and the loss of enzyme recognition sites after...BpiI/BbsI to efficiently assemble up to six DNA fragments at a time. As with all Golden Gate-based methods...outside their recognition site and permits DNA fragments with compatible overhangs to be efficiently assembled...

...site-specific recombination events. The system consists of two components derived from the P1 bacteriophage...present; intramolecular events happen with greater frequency than intermolecular events, but any two sites ...series, we examined a number of important plasmid elements – promoters, origins of replication, protein tags...recognition site. The P1 bacteriophage uses these components as part of its natural viral life-cycle, and ...and researchers have adapted the components for use in genome manipulation. Cre recombinase, originally...allowing for broad applications in a variety of experiments. LoxP (locus of X(cross)-over in P1) sites are...modification can be restricted based on experimental constraints. This has been used for a wide range of purposes...

...PAM sequence of NGG (where N represents any base), their engineered variants are more permissive: SpG can... and mouse model systems, testing base editing variants, and assessing the safety of their intervention... al., 2020).  Five years later, three of these variants (ABE8.8, ABE8.20, and ABE8e) would be tested for...problem, Benjamin Kleinstiver’s lab engineered new variants of S. pyogenes Cas9. While the wild-type enzyme...in cellular models tested both the SpG and SpRY variants of Cas9. With many PAMs available, a large number...the most efficient editing. In these initial experiments, SpG Cas9 came out on top. Narrowing PAM preferences...worked on the opposite project: engineering Cas9 variants with a preference for specific PAMs. Though near-PAMless...

...the suggested amount, instead of the other dry ingredients above. Loosely close the cap on the bottle (do...

...each level, as each level has different safety requirements. BSL-1 is designated for those working with ...

...money on optimizing experiments, to false conclusions.  Figure 1. Potential points where DNA errors...shifts, missing elements, and nucleotide indels or mismatches in significant plasmid elements. Which differences...an important part of maintaining the quality of reagents, especially shared ones.  The importance of QC... propagation of plasmids in bacteria — has many points at which small errors can be introduced. Eventually...introduced, indicated by exclamation marks. Error points include places where DNA is being created, such...tags, promoters, and other critical functional components. Fun fact! Some older plasmids in our collection...each of their plasmids, from descriptions of key components of the plasmids to partial, full, or annotated...

...link together multiple fragments, allowing ordered assembly of multiple components into a destination vector...suitable for other model systems, including E. coli, plants, Drosophila, and zebrafish!   Why use multiplexed...(here, O1-4) adjacent to the cut sites “link” fragments together and drive ordered assembly into a Cas9...and thus the highest frequency of genome editing events. Yamamoto Lab Multiplex CRISPR/Cas9 Assembly Kit... with the Gersbach lab plasmids, multiple Cas9 variants are available: wt humanized Cas9, D10A nickase...clone your own entry vectors containing other Cas9 variants to use with the MuLE system. Multiplexing from...are also available from the Yang lab for use in plants. The mammalian multiplex systems use the Csy4 RNA...

...kit components Recyclable Yes, all kit components and packaging Yes, most kit components and... preps, while other applications require large amounts of high quality DNA. Figure 1 illustrates the three...Bacterial transformation protocols require such small amounts of DNA (often on the picogram scale) that most ...highest-competency cells you will still struggle to get transformants if the plasmid you are using is completely degraded...mammalian cell transfections can require quite large amounts of DNA (easily in the micrograms scale and up)....more importantly, the DNA needs to be free of contaminants, such as bacterial endotoxins, that could affect... Another is the possibility of carrying over contaminants from the plasmid prep process into the final...

...can vary "Lab assistants" usually wash glassware, prep media, etc. "Research assistants" are more involved... researchers, as well as graduate students (your teaching assistants), about their experiences. Research...investigator (PI) Runs the lab Writes a lot of grants Varying degrees of engagement with what happens...degree "Rotation student" - some PhD programs have students rotate through a few labs, ~10 weeks per lab, ...way to answer those questions. I’ve met graduate students who enjoy the thrill of acquiring a piece of knowledge...with your labmates and mentors Many of the grad students and postdocs I’ve met have done things that I ...about careers in science are geared towards grad students and postdocs—people who are a little ahead along...

...assembly, assembling fragments in multiple steps to avoid using multiple fragments that share homology ...cloning method, but it is limited by its sequence constraints. The long overhangs must not contain the dNTP...ligation-independent cloning (SLIC), eliminated many of LIC’s constraints by relying on the power of homologous recombination...the linearized vector by T4 digestion; then the components are combined and annealed. The resulting recombination...up a new world of multicomponent assembly, as fragments with overlapping sequence homology can be assembled...of multicomponent assembly with SLIC. Multiple fragments are PCR amplified to add 5′ and 3′ homology regions...vector. 5′ overhangs are generated on all the fragments and the vector, specifying the order of assembly...

...few Plasmids 101 posts, we focused mainly on the elements required for plasmid maintenance within an E. ...different cell types and each one requires different elements for vector propagation. This post, along with ...and explain how they differ from the bacterial elements previously described. Why do scientists use yeast...complex internal cell structures similar to those of plants and animals. Unlike bacteria, yeast can post-translationally...in the lab typically contain all the necessary components to allow shuttling between E. coli and yeast ...the bacterial cell. Similarly, the specific ORI elements included within a yeast vector determine how the...replicated and maintained within the yeast cell. These elements control not only the number of plasmids found ...

...functions. Experiments come next, testing the correctness of the hypothesis. The results of experiments can ...already encouraged by “Broader Impacts” requirements for certain grants) (28). As one young scientist, Matthew...Politics and science are intrinsically linked: governments exist in reality and science is how we learn ... to protect their rights (2). Less formally, governments are the organizations that make and enforce the...information about reality informing people and their governments on what outcomes are actually possible for a ...informed fashion. For science to be useful to governments, however, inconvenient data must not be ignored...climate change page on whitehouse.gov, freezing grants, and proposing to defund aspects of research at...

...the FP(11) and FP(1–10) fragments. When Protein A and B interact, the FP fragments can assemble the full...al. 2017 under CC-BY license. Many split-FP fragments are named according to the number of strands in...10 and 11 to yield large (1–10) and small (11) fragments. In several cases an FP(1–10) fragment may be ...be compatible with multiple FP(11) fragments, even from a different base FP (for example, EBFP2(1–10) can...Venus Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis...pBiFC-VC155 Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis...Chang-Deng Hu mVenus pET-BiFC contains both fragments of split mVenus (aa 155), includes I152L in N-...

...PAM with no sequence constraints, or at least has looser or different requirements. Luckily, there are ...most gene targeting experiments. Between SpRY and several of the other Cas variants described, nearly all...need for gene editing in research, the old PAM constraints just won’t cut it anymore. Say hello to PAM-free...PAM must be selected. When it comes to CRISPR experiments, therefore, you often must make a choice about...region will do the job. What matters for these experiments is cutting efficiency so that the greatest effect...that don’t utilize NGG   There are plenty of Cas9 variants which utilize alternative PAMs. These alternative... generating Cas enzymes with alternative PAM requirements (Zhao et. al, 2023). xCas9 has been shown to...

... the Rare Genomics Institute to provide plasmid grants to researchers working on rare diseases through...group recently reviewed and reported that PMM2-CDG patients suffer from immunological defects. These are characterized...almost every aspect of immunology, participating in events such as cell development, recognition and migration...migration, the immune compromise observed in CDG patients is not surprising. However, the molecular and cellular...cellular mechanisms behind the immune defects in CDG patients are still poorly studied and understood.  In line... to lower the burden of immune symptoms on CDG patients and to increase their quality of life by reducing... the lab will be greatly facilitated by CRISPR reagents and other specific gene sequences available from...

...CRISPR Affinity Purification in situ of Regulatory Elements) method. The original CAPTURE method was published...CRISPR Affinity Purification in situ of Regulatory Elements: The original CAPTURE method The original CAPTURE...of a stable cell line that co-expresses three components: dCas9 with an added biotin acceptor site BirA...genomic location of interest  When these three components are co-expressed, the gRNA targets the dCas9 ...then developed by detecting these chimeric DNA fragments by PCR or next-generation sequencing. Figure...diagram). Cross-links are reversed so that DNA fragments can be detected by PCR or deep sequencing. ...number of cells (~5x107). Together, these two requirements prevent the use of CAPTURE with primary cells...

...organizing events. I contacted local professional organizations and nonprofits. I volunteered at events like...resources daily to promote CINSO events and recruit speakers and participants for our professional development...Science Outreach, or CINSO. We organize fun science events for adults and professional development opportunities...best I began viewing my activities as types of experiments. I searched for new opportunities with an open...wasn’t something I liked – well, I was used to experiments not working, so just like in lab, I sucked it... – call it “troubleshooting.” Trying out new experiments and new ideas is nothing new to a scientist. ... opportunities provided by your programs and departments Graduate school was an excellent time to try ...