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We narrowed to 1,020 results for: NTS;

Showing: 261 - 280 of 1020 results

  1. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

    Type
    Blog Post
    ...many microscopy experiments with fluorescent proteins (FPs) localized to compartments other than the cytoplasm...misfolded FP-fusions on quantitative imaging experiments and cell physiology in general. Pitfalls of Fluorescent...apparent and can confound quantitative imaging experiments or even negatively impact cells (see Figure 1...sequences, which often have absolute position requirements. For example, a KDEL ER retrieval sequence only...that FPs are often poorly suited for cellular compartments other than the cytoplasm. Generally, FPs evolved...in or were engineered for use in cytoplasmic environments. However, approximately 40% of human (and most... localize to chemically distinct subcellular environments, including the organelles that compose the secretory...

  2. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
    ...conditions. Purifying DNA fragments Once you’ve generated your DNA fragments, it is always a good habit...digested fragments or PCR products from a gel. You will avoid contamination from other DNA fragments and you...nanodrop to assemble the  fragments based on their molar ratio. Assembly of the fragments There are multiple ... way to do it (i.e. avoid assembling too many fragments at once, if it is possible). There are many softwares...could affect or delay your experimental work. Fragments generation After you’ve designed your cloning ...cloning scheme, you’re now ready to generate fragments. You can generate the parts of DNA that you want to assemble...between the different enzymes. Always check the fragments size of your digestion on an agarose gel. If the...

  3. Viral Vectors 101: Calcium Sensors

    Type
    Blog Post
    ...neuron subtype) and specific subcellular compartments for measurements. All of the sensors discussed below ... you are measuring the events-of-interest. Sensors suitable for ER measurements include: CEPIA (additional...are used to monitor calcium concentrations and transients in living cells and organisms. Unlike chemical...slower). GCaMPf may be suitable for very rapid measurements, like action potentials in neurons. jGCaMP7 ...calcium concentrations in different subcellular compartments can vary greatly. Additionally, the calcium ... the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) subsequent change in...you combining your calcium reading with other measurements, specifically fluorescent ones? Many of the ...

  4. Hot Plasmids: Winter 2025

    Type
    Blog Post
    ...uniquely challenging to apply in dicotyledonous plants, or dicots. But Jae-Yean Kim’s lab recently "broke... strategy uses new combinations of PE protein components (Figure 1), an altered epegRNA delivered with...chaperone, RT: reverse transcriptase. Right: T1 tomato plants that inherited an edited gene to confer resistance..., two weeks after herbicide was applied to both plants. Adapted from Vu et al., 2024 bioRxiv, under CC-BY-NC-ND...both tomatoes and Arabidopsis, suggesting the improvements may be broadly applicable to dicot species. .... In addition, they generated offspring tomato plants that inherited the genomic edit but not the editing...desired edits in tomato and Arabidopsis. Nature Plants, 10(10), 1502–1513. doi: https://doi.org/10.1038...

  5. Quantifying DNA? Here are Five DNA Quantification Methods to Consider

    Type
    Blog Post
    ...electrophoresis. Smaller fragments migrate quicker than larger fragments. These fragments are quantified over...the DNA such that smaller fragments are quantified first before larger fragments. Figure 1: The quantification...units over time. Smaller fragments migrate more quickly than larger fragments. Image from OpenWetWare....your DNA prep and whether there are any other contaminants. Nucleic acids (DNA and RNA) absorb maximally...quick and simple and doesn’t require any special reagents. However, there’s limited sensitivity at low concentrations...piece of DNA. This method is best used for DNA fragments (like a PCR product). This method also gives an...a few minutes per sample. During the run, DNA fragments move through a micro- or nanofluidic channel and...

  6. Antibodies 101: Reading a Flow Plot

    Type
    Blog Post
    ...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...

  7. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    ... 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...

  8. Choosing the B(right)est Fluorescent Protein: Photostability

    Type
    Blog Post
    ...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 ...

  9. Plasmids 101: Golden Gate Cloning

    Type
    Blog Post
    ...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...

  10. Plasmids 101: Cre-lox

    Type
    Blog Post
    ...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...

  11. The Advances Behind The World's First Personalized CRISPR Treatment

    Type
    Blog Post
    ...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...

  12. A Look at Addgene's QC Process

    Type
    Blog Post
    ...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...

  13. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    ...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...

  14. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    ...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...

  17. A Guide to Getting Started in Undergrad Research

    Type
    Blog Post
    ...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...

  18. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

    Type
    Blog Post
    ...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...

  19. Plasmids 101: Yeast Vectors

    Type
    Blog Post
    ...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 ...

  20. March for Science

    Type
    Blog Post
    ...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...
Showing: 261 - 280 of 1020 results