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We narrowed to 1,025 results for: URE

Showing: 181 - 210 of 1025 results

  1. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    ... efficiencies using sequence features and DNA repair determinants. Nature Biotechnology 2023, 1–11. https... evaluate all of the pegRNAs in one experiment (Figure 1). Through this method, we found key determinants...Once we had our datasets with all the different features and editing efficiencies, we could train our prediction...very diverse edit types and cell contexts. Figure 1: High-throughput screening of pegRNA efficiency...downstream flanking sequences to their input string (Figure 2). This string can then be used as input for the...e.g., top 5) and test them in their labs. Figure 2: Guidelines to predict pegRNA efficiencies with...Mathis et al., 2024) and concurrent research by the Shendure lab (Li et al., 2024) have highlighted how chromatin...

  2. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    ... SDS-PAGE, measure the protein content and normalize to ensure equivalent loading. Denature the samples...antibodies.   Figure 1: Overview of the Western blot process. The sample, a mixture of protein is separated...denaturing western, the protein is denatured to its primary structure and separated by size with smaller...western, the protein’s secondary and tertiary structures remain intact and the protein is separated through... expensive system-specific sandwiches.   Figure 2: The western blot dry apparatus includes a stack...its native state while a western blot detects denatured polypeptide chains. The protein regions available... antibody validation. This level of validation ensures that the antibody is staining the intended target...

  3. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    ...without DNA cleavage. Nature, 551(7681), 464–471. https://doi.org/10.1038/nature24644 Gaudelli, N. M., Lam...double-stranded DNA cleavage. Nature, 533(7603), 420–424. https://doi.org/10.1038/nature17946 Komor, A. C., Zhao,...other or one pyrimidine base to the other. Figure 1: Base transition editors catalyze purine-to-purine...strand as a template, copying the base edit. Figure 2: Cytidine deamination takes place on the free...base editing efficiency for mammalian edits is to ensure the editors make it into the nucleus and that they...and specific CRISPR-based cytosine base editing. Nature Biotechnology, 41(5), 663–672. https://doi.org/...increased activity and therapeutic application. Nature Biotechnology, 38(7), 892–900. https://doi.org/...

  4. An Inside Look into Addgene's Dev Team

    Type
    Blog Post
    ...of Addgene’s work culture. Our developers are a diverse group of ethnicities, cultures, genders, educational... for working on a new feature. We start with a story that describes the feature and what the criteria ...stakeholder accepts the new feature as done and we’re onto the next story. Figure 1: Addgene’s Agile Approach...working on a feature, sometimes we have to take short cuts or add a band-aid to get a feature to work. We...number of stories we commit to for each sprint, to ensure we will have the capacity to finish all of the ...criteria is for accepting the feature as done correctly. These initial stories are usually a result of collaborations...step-by-step instructions on how to demonstrate the feature. For example, we recently reduced the number of...

  5. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    ...sequence (Zong et al., 2018) (see CBE-3 in Figure 3). Figure 3: Activity windows of plant base editors...excision repair mediated by uracil glycosylase (Figure 1). The presence of a uracil glycosylase inhibitor...generation of the desired T-A base pair. Figure 1: Transition mutation (C>T, G>A, A>G, and T>C)... that compete with the desired outcome are not pictured. Adenine base editors (ABE) What if you have...satisfactorily accepts DNA as its substrate (see figure 2).          The artificially evolved adenosine...genomic A-T base pair is converted to a G-C pair (Figure 1). As the cellular inosine excision repair is ...base editing platforms for plant systems.  Figure 2: Plant base editor platforms and their molecular...

  6. Plasmids 101: E. coli Strains for Protein Expression

    Type
    Blog Post
    ...better at 30°C or room-temperature, which is accomplished by growing your culture to the desired density...previous Plasmids 101 blog, we reviewed the salient features of several popular strains of E. coli for DNA ...except ** which are K12 Strain Resistance Key Features Genotype Use BL21 (DE3)   Basic IPTG-inducible...Denotes the presence of an additional plasmid-- make sure to maintain this by growing on appropriate media...timing is important, as you typically want to make sure your cells have first reached an appropriate density...expression. If you wait too long to induce, your culture will start collecting dead cells, and, conversely...too early as there are not enough cells in the culture to make protein.  The DE3 lysogen/T7 promoter combination...

  7. Tips for Using FRET in Your Experiments

    Type
    Blog Post
    ...acceptor fluorophore can be measured using microscopy techniques. FRET measurement sensitivity makes it suitable...one of the first techniques which enabled the measurement of single molecule interactions in living cells...living cells. Since then FRET has also been used to measure conformational changes, cleavage activity using...is still one of the best and most used pairs to measure FRET. The table below lists plasmids that can be...and Venus Other issues that can affect FRET measurements include: the brightness of a fluorophore pair...between FRET efficiency and cross-talk. Methods to Measure FRET for Cell Biology Studies Several methods have... have been used over the past 20 years to measure FRET and there is not one that is better than another...

  8. Developing a cold tolerant E. coli using specialized chaperones

    Type
    Blog Post
    ...environments: corrosive pH ranges, crushing pressures, near-melting temperatures, tons of radiation, or freezing ...Sreepadmanabh M from IISER Bhopal. All illustrations / figures featured were designed by our team member Kallol Mozumdar... take bacteria out of their optimally suited temperature ranges and make them grow better at suboptimal...cold-loving) bacteria. These bacteria inhabit cold temperature locales and psychrophilic enzymes help them survive...next-door microbes which grow optimally in moderate temperatures (usually defined between a fairly nippy 20°C...materials degrade at a much slower rate under cold temperatures, and frigid conditions decrease the potential...mesophilic host systems. The so-called “regular” temperatures of growth, say around 37°C, is a far cry from...

  9. Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

    Type
    Blog Post
    ...method does provide a quantitative measure of autophagy, it only measures bulk autophagy (as opposed to giving... Long-lived protein degradation: This approach measures the degradation rate of radio-labeled long-lived...long-lived proteins as a proxy for autophagy. Cells are cultured with isotope-labeled amino acids for several hours...by measuring the amount of radioactivity in the culture supernatant (this is indicative of protein degradation...out of this assay. This is also a quantitative measure of autophagy, but, like the protein degradation...degradation assay above, it’s slow and only measures bulk autophagy. Morphological assays Electron microscopy...method relies on the identification of autophagic structures based on morphology. Autophagosomes are relatively...

  10. Replacing Paper: Tips for Choosing an Electronic lab Notebook

    Type
    Blog Post
    ...articles, only 46% could be reproduced. In a recent Nature report from researchers at Amgen, it was stated...that only 11% of the academic research in the literature was reproducible by their groups. Finally results..., it will always be a part of everyday work to capture ideas, write down certain notes or brainstorm, ...functionalities i.e. main priorities of different ELN features, ranging from whether respondents saw them as ...term, so they need a solution for which they are sure that there will be no sharp changes in costs once...researchers to store everything in one place. From pictures to excel sheets, researchers today generate vast...Following these findings, we defined the priorities for future ELN development and developed the SciNote Electronic...

  11. Typing CRISPR Systems

    Type
    Blog Post
    ...with CRISPR–Cas13. Nature, 550(7675), 280–284. https://doi.org/10.1038/nature24049 Altae-Tran, H., Kannan... ideas for your own research! Classification Structure Principles of CRISPR classification While CRISPR...six types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy...using a Cascade complex is tricky, as you need to ensure every unit of the complex expresses at similar ...components present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits...CRISPR types do, due to a lack of canonical CRISPR features (Pinilla-Redondo et al., 2019; Moya-Beltrán et... most often found on plasmids. The many unique features of IV-A and IV-B have led to the hypotheses that...

  12. Early Career Researcher Toolbox: Finding Relevant Papers

    Type
    Blog Post
    ...analyzes publications and figures using machine learning to extract important features such as influential ... top of the ever expanding body of biomedical literature! PubMed PubMed is the classic search engine ...eventually replace the existing version of PubMed.  Key features This new version has an improved search algorithm...updated. This Twitter list contains >300 Twitter literature bots, including a bot for biosensor papers, microbe...Semantic Scholar Semantic Scholar is an AI-backed literature search developed by the Allen Institute for Artificial...that are more relevant to your search terms. Key features Semantic Scholar has several ways to focus your... populated at the top of this filter. A unique feature of Semantic Scholar search results are the topic...

  13. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    ...to compare exposure times of our samples. A prominent, strong signal has a shorter exposure time than ... be essential to your new research project, but unsure which one will mark your target the best, or even...The strength is based on binding specificity and exposure time. We first have a negative control which does...align with what the atlases (mentioned above) and literature suggest, providing an expectation and comparison...imaged pattern does NOT align with the atlases/literature and is unrelated to the target protein, we categorize... goldilocks antibody selection process The exposure time of the primary antibody compared to negative...control also determines the signal’s strength. Exposure time is the amount of time the sample needs to...

  14. Viral Vectors 101: Calcium Sensors

    Type
    Blog Post
    ...noise and ensure you are measuring the events-of-interest. Sensors suitable for ER measurements include:... requiring only a simple measurement. However, single wavelength measurements are more susceptible to ... RE. Structure-and mechanism-guided design of single fluorescent protein-based biosensors. Nature chemical...subtype) and specific subcellular compartments for measurements. All of the sensors discussed below fall into...slower). GCaMPf may be suitable for very rapid measurements, like action potentials in neurons. jGCaMP7 ...additional versions are available to simultaneously measure calcium changes in the ER, cytosol and mitochondria...you combining your calcium reading with other measurements, specifically fluorescent ones? Many of the ...

  15. Cloning Mammalian Cells with the Agarose Method

    Type
    Blog Post
    ...using cloning rings. A picture of a 60 mm dish ready for picking is shown in figure 1.   Find Additional... Protocol Pages Agarose cloning method procedure The procedure (illustrated in the YouTube video below...well plate is also prepared with about 200 ul of culture medium containing serum in each well. A 200 ul ...both Hepes and bicarbonate) is brought to room temperature and placed in the hood. The medium from a 10 ...is capped, swirled and tested for the correct temperature by placing on the wrist or cheek. Within less...warm but not hot- and still liquid- it is rapidly poured onto the middle of the plate, where it immediately...the agarose cloning method Test your agarose temperature. Cloning cells using an agarose overlay does ...

  16. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    ...editors for more details on how they work!) Figure 1: Base transitions are edits between the purine...side groups of DNA bases and leave the core ring structure intact, allowing them to make 4 out of the possible... Cas9 are sequence specificity and the R-loop structure. In order to bind DNA, Cas9 requires (1) a guide... a region of separate strands called an R-loop (Figure 2). This allows the guide RNA to base pair with...accessible on the outside of the enzyme. Figure 2: CRISPR R-loop. Created with BioRender.com. ...abasic sites with A's, producing C → A edits. Figure 3: Mechanism of cytosine transversion editors. ...experimented with adding different proteins to the CGBE architecture. They conducted a CRISPRi screen to identify...

  17. New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor

    Type
    Blog Post
    ...kinetics which are illustrated in figures 2 and 3.  Figure 2: Glutamate titration curves for ...from Aggarwal et al…, Supplementary Figure 3.   Figure 3: pH titration curves and ∆F/F0 for...recommend using the SGZ for in-culture work only, GPI anchors for in-culture work and 2P in vivo imaging,...using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation and emission spectra of...iGluSnFR3 v857. Image adapted from Aggarwal et al., Figure 1   Increased versatility through two different...Image adapted from Aggarwal et al…, Supplementary Figure 2.   Improved trafficking and localization... constructs. Image adapted from Aggarwal et al… Figure 4. PDGFR is a C-terminal fusion to the PDGFR transmembrane...

  18. 5 Great Apps for Lab Life

    Type
    Blog Post
    ...content we store in them: music, emails, chats, pictures, and apps. Throughout my time as a graduate student...allowing you to upload anything from protocols to pictures of gels or even sketches to your notebook. The...saving trees! The Earth thanks you. 2. Gibco Cell Culture by Gibco Whenever I start to learn a new technique...I digress. When I started learning proper cell culture technique, I tried to find an app that could help...calculations. This is how I came across the Gibco Cell Culture app. Gibco organizes its app into four categories...are a lifesaver when your research requires cell culture and include a counter, lab timer, dilution calculator...calculator, and a couple of other nice features. This app also has a great educational section. This section...

  19. Hot Plasmids: Fall 2024

    Type
    Blog Post
    ...solubility and stability (Figure 2).      Figure 2: Crystal structures of CRBNmidi alone (A) and...establishing CRBNmidi as a promising tool for future structure-guided drug design and studies of protein ...domains and is recommended for use in western blots (Figure 1). If you prefer to produce your own antibody ...chain are also available from Addgene!   Figure 1: Experimental validation for Anti-V5 [IPI-SV5...dimmer, showed greater photostability and also matured considerably faster, with expression levels comparable...comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also offered clearer signals due to...autofluorescent tissues like the intestine. Figure 3: Expression of fluorophore-tagged GOLG-4 through...

  20. Twenty Years of Sharing: Addgene's Viral Vector Service

    Type
    Blog Post
    ...growing in requests faster than jGCaMP7 (Figure 5B).   Figure 5: A) GCaMP6, jGCaMP7, and jGCaMP8 viral...setup. Teams across Addgene have worked hard to ensure that the service has grown steadily, helped by ...our catalog had grown to 879 viral catalog items (Figure 1). The service caught on quickly in the scientific...distributed over 100,000 viral vector preps!   Figure 1: A) Cumulative AAV preps available in the Addgene... they’re delivering their payload to.   Figure 2: Numbers of AAV tools, by category, available...respectively, by substrates, light, or engineered ligands (Figure 2). Biosensors, which include calcium and voltage...explain why they are the most commonly requested (Figure 3). Over 400 biosensors were distributed in 2023...

  21. Neuronal labeling with Spaghetti Monster

    Type
    Blog Post
    ...neuronal sub-cellular structures GFP is limited in its ability to label subcellular structures in the CNS. GFP...connections (Shepherd, 2004). Understanding the structure and function of these neural circuits is essential...experiments and visualize numerous molecules or structures at once (Shaner, 2007). In contrast to epitope...for the cells and affects the labeling of small structures like neurites. Like epitope tags, low FP expression...church of the Flying Spaghetti Monster; the smFP structure resembles images of the deity (see comparison ...processes. In contrast, smFPs can label fine neuronal structures with high fidelity and at lower concentrations...GFP. smFPs enable better labeling of these fine structures through their multiple, high-affinity binding...

  22. AAV Titers: Where Do They Come From And What Do They Mean?

    Type
    Blog Post
    ...preparations are genome-containing (Figure 1).    Figure 1: Electron micrograph of an Addgene... by the temperature parameters. For example, we found that changing the annealing temperature of the PCR...viral vector solution, and generally these methods measure different characteristics of the solution. These... containing viral genomes. Physical titers are measured by quantifying the concentration of viral genomes... report the physical titer of a viral solution (Figure 2). Because physical titers are used for dosing...values mean and how they can be compared.   Figure 2: Label that comes on Addgene’s AAV aliquots. ...circled in red) report physical titer, which is measured by qPCR.   Physical titer is typically calculated...

  23. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    ... of temperature on total crop production and the production window. A two-way MANOVA could measure the..., you could test the impact of temperature, humidity, sunlight exposure time, and watering volume on total...power. Not sure if your data meets the assumptions of parametric tests? If you aren’t sure if your variance...outliers or data spread within those data sets. Measures of dispersion – standard deviation/error, variance...in a certain direction (i.e., you just want to measure if a sample is greater than a control), then a ...variable. A two-way ANOVA could test the effect of temperature and humidity on an outcome such as total crop... one-way ANOVA can test either the effect of temperature or humidity on crop production.   MANOVA The ...

  24. Live and Let Dye: Self-Labeling Protein Tags

    Type
    Blog Post
    ...applications.   Figure 1: Live-cell labeling strategies. The green structure represents a fluorophore... end up linked to whatever molecule you choose (Figure 1). Just express the fusion protein and add a specific...fluorophore or other useful ligand, gray structure is the protein of interest, and black is the fusion tag ...-tag construct can help you better detect dim structures or low levels of expression with the same imaging...super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and... enables applications like multi-color imaging (Figure 2). If you’re working with purified proteins in...colors depending on the needs of your experiment (Figure 3). And using self-labeling tags makes updating...

  25. Sequencing Options for CRISPR Genotyping

    Type
    Blog Post
    ... Figure 1: Schematic representations of the indicated unbiased genotyping options. Figure reproduced...other elements, including those relating to DNA structure (e.g. epigenetic modification, bulges) that may...conventional algorithms may not provide a full picture of the impact of CRISPR editing in the model cell...negatives Combining sequencing techniques can ensure validated experiments Unbiased detection methods...sequencing and in silico prediction should give a broad picture of all possible editing events in the genome; from...induced by CRISPR-Cas nucleases in human cells." Nature biotechnology 31.9 (2013): 822-826. PubMed PMID...reveals RNA-programmed Cas9 nuclease specificity." Nature biotechnology 31.9 (2013): 839-843. PubMed PMID...

  26. Flow Cytometry Readouts: Yes, No, and Everything in Between

    Type
    Blog Post
    ...downregulation. Figure 3 shows a histogram for a yes-no dot plot, like the one shown in Figure 1.  ...populations in the histogram plot in Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes...Before you read out your plots, you’ll want to make sure you’ve also adjusted voltage and compensated your...adjusting voltage: During data acquisition, make sure that the signal for the positive population is no...should overlap with the negative control population (Figure 1). Based on where the GFP- population ends, you... the total cells present in the gate.   Figure 1: Example of yes-no flow plot of the (negative...expression, low expression, and everything in between (Figure 2). These smears tend to occur when you are looking...

  27. Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens

    Type
    Blog Post
    ...Nishimasu H, Nureki O, Zhang F. Nature. 2015 Jan 29;517(7536):583-8. doi: 10.1038/nature14136. Epub 2014...Cas9’s crystal structure. “I thought maybe we could use information from the structure to guide and improve...technology. These libraries are lentiviral in nature, so please ensure that you are equipped and authorized to...genes, it is not possible to test each one and make sure it’s working. You can only do that screen with a... the SAM pooled library In a paper published in Nature in December, Konermann and her colleagues in Feng...-scale, knockout screening). As the Zhang lab's Nature paper reports, “Here we have shown that the SAM...Therapeutic uses in humans are also on the table. “In the future, there is potential there,” he says. "We have to...

  28. Plasmid Preps: Different Purity, Different Quantities, Different Uses

    Type
    Blog Post
    ...the purification procedure. Good indicators of DNA purity are the absorbance measurements at 230, 260, and...same. Before purifying a plasmid from a bacterial culture, it is important to consider your experiment.  ...Cloning grade DNA Transfection grade DNA Figure 1: Transformation introduces transformation grade...range). It can be extracted from small bacterial cultures (i.e. 2-3 ml) with or without commercially available...and DNA for transformation doesn’t need to be as pure as for other uses.   Cloning grade DNA Molecular...also dictate the required sizes of the bacterial cultures and commercial kits you will adopt for the extraction... cloning it is important that the preparation is pure from other nucleic acids (genomic DNA and RNA), ...

  29. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    ...unique changes in their spectral properties upon exposure to a specific wavelength of light. PA-FPs can ...fluorescence, to a brighter fluorescent state by exposure to a specific wavelength of light. The switch ...switches from green to red fluorescence. mEosFP exposure to UV light causes an irreversible cleavage near...formation of mEosFP’s chromophore requires a temperature below 30°C and this is not ideal for experiments...emission of green fluorescence, but then further exposure to blue light also inactivates the chromophore...state. Dronpa can be reversibly reactivated by exposure to UV light. The fact that blue light both activates...photons are being emitted, making it easier to capture a clear image above background. The majority of...

  30. Illuminating Choices: A Guide to Selecting Fluorescent Dyes and Ligands

    Type
    Blog Post
    ... of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes, and... fluorescent labels. In Nature Methods (Vol. 19, Issue 2, pp. 149–158). Nature Research. https://doi.org...visualization of both organic tissues and cell cultures, opening biological interiors to many inquisitive... and fluorescent ligands. Figure created with biorender.com. Fluorescence refers to the emission ...Fluorescent dyes and ligands vary in their chemical structure, leading to differences in their excitation and...ability to retain fluorescent intensity during exposure to light, is another factor to consider. If your...your application requires longer periods of exposure, you’ll want a more photostable fluorescent protein...
Showing: 181 - 210 of 1025 results