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We narrowed to 1,025 results for: URE

Showing: 211 - 240 of 1025 results

  1. A Look at Addgene's QC Process

    Type
    Blog Post
    ... to ensure all plasmids pass our high-quality standards and contain the expected plasmid features.  ...based on SnapGene’s “Common Feature” library — with some additional features added by the QC team — and...the plasmid to ensure our assembly result is accurate The gene/insert or any major feature highlighted by...Nobody’s perfect! This is a platitude I’m sure we have all heard. It’s a truth that extends to both people...optimizing experiments, to false conclusions.  Figure 1. Potential points where DNA errors can be introduced... Figure 2. Addgene’s basic QC process. Created with BioRender.com. Once Addgene receives deposited...resolving a variety of QC issues. This process ensures that any potential sequence discrepancies can be...

  2. Viral Vectors 101: Virus Safety

    Type
    Blog Post
    ... alert the day of the procedure. Mice move much more quickly than cell cultures do, and tired researchers... some nerves. Following safety procedures and practicing safety culture, however, can let you work with...risks and how to minimize them. Built-in safety features   When viruses were first being engineered as ...still enter (and thus infect) cells, this safety feature prevents the virus from replicating and spreading... including yourself as the researcher.   Figure 1: Separation of retroviral packaging components...precautions to take, and if something does happen, ensure you can receive appropriate treatment and/or monitoring...monitoring. Virus-specific concerns   In nature, some viruses are more dangerous than others, and the...

  3. Antibodies 101: Flow Compensation

    Type
    Blog Post
    ...present in the sample (APC in the case of Figure 1). In Figure 1A, the population needs to be shifted towards.... Let's look at an experimental example in Figure 2. Figure 2A shows the sample prior to compensation....the populations move, as in Figure 2, following compensation. Figure 3: Flow plot for a double positive... a compensation value between the two channels (Figure 1A). Applying compensation means you are telling...values later in your analysis, unlike voltages.  Figure 1A shows a single-color APC control, with APC and... a straight line and are only positive for APC (Figure 1B). For single-color samples, you can only change...would decrease the compensation value.   Figure 1: Compensation example with single color control...

  4. Polymerase Chain Reaction Overview and Applications

    Type
    Blog Post
    ...also stable at this temperature and can bind to the primers.  Extension- The temperature is then raised slightly...slightly to Taq polymerase’s ideal temperature (70-75oC). At this temperature Taq polymerase can synthesize...and the use of a fluorometer feature built into the thermocycler to measure that fluorescent output. With...resistant enzyme that can withstand changes in temperature. Taq was first identified in the late 1960s during...separation of the two DNA strands.  Annealing- The temperature is then dropped to a range in which the forward...forward and reverse primers are stable. At this temperature the primers can anneal to the single stranded...the target DNA quickly and accurately.  Figure 1: Steps in PCR reaction   This process of ...

  5. Early Career Researcher Toolbox: Free Tools for Making Scientific Graphics

    Type
    Blog Post
    ...your own figures Google Drawings Google Drawings is similar to using PowerPoint to draw figures. It’s part...useful for drawing figures with lots of elements. It took some playing around to figure out all the settings...project my team & I have poured our hearts into for 2 yrs) has 200K+ figures by 95K+ scientists 👨🏽‍🔬...when writing posts. Since then I’ve realized my figures are just as important, if not more important, than...drawing tools, so it’s harder to draw intricate figures, like a brain or a mouse. Vectr Vectr is like a... Vectr to be fairly intuitive and I was making figures quickly. Inkscape Inkscape is the most similar...Illustrator out of the three options for drawing your own figure discussed in this post. It’s open source and available...

  6. The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

    Type
    Blog Post
    ...design goals. Examples:  “feature aware”, “SNP aware”, “secondary structure aware”, and “microhomology...collective utility, it’s easy to fault the lack of features in a single application. The question is then,... solve, and finally how we might proceed in the future. When we think of designing experiments using CRISPR...have broken down the tools into the individual features that make each tool unique. The CRISPR Software...Software Matchmaker is composed of these features and enables you to select the tool(s) based on your project...advanced functionality, it seems these kinds of features are delegated to other more suitable software ... removing repetitive tasks and/or by providing features to help with the post-design process such as primer...

  7. FLEx Technology and Optogenetics: Flipping the switch on gene expression with high spatial and temporal resolution

    Type
    Blog Post
    ...activates halorhodopsins to silence neuronal activity (Figure 1) (Wiegert et al., 2017). How to get an opsin...into the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable...promoter only in specific neuronal cells. FLEx ensures, therefore, spatial accuracy and strong opsin expression... functional topography of the neural circuit architecture of the brain regions associated with motivated...on developmental biology at the École Normale Supérieure de Lyon. Her love for educational science led...genetically targeted optical control of neural activity." Nature neuroscience 8.9 (2005): 1263. PubMed PMID: 16116447...: PMC2820367. Deisseroth, Karl. "Optogenetics." Nature methods 8.1 (2011): 26. PubMed PMID: 21191368. ...

  8. Hot Plasmids - November 2023

    Type
    Blog Post
    ... and in vitro applications.   Figure 2: Modular features of DIRECTED particles where the fusion...-the-art. PE6b was very efficient for short, unstructured templates and significantly reduced undesired...efficient at complex edits, such as long or highly structured templates. Cas9 variants PE6e–g often had even...varied depending on the specific edit.   Figure 1: Choosing the best PE6 variant for your application...type-specific delivery by modular envelope design. Nature Communications, 14(1), 5141. https://doi.org/10.1038...therefore been trafficked between locations). Figure 3: A) LOV-Turbo becomes active when illuminated... control of proximity labeling in living cells. Nature Methods, 20(6), 908–917. doi: 10.1038/s41592-023...

  9. Plasmids 101: Codon usage bias

    Type
    Blog Post
    ...codon optimality reveals hidden signatures of cotranslational folding." Nature structural & molecular biology20.2...but some possible reasons include: Metabolic pressures - it takes cellular resources to produce tRNAs...Protein folding - If a protein is encoded by a mixture of codons with highly and poorly charged tRNAs,...codons are associated with specific secondary structures in 10 closely related yeast strains. Adaptation...properly. You might then think, “Okay, I’ll just make sure the abundances of the codons I choose for the host...Angov et al., 2008), but there are still many more features to consider when designing a full gene. A non-...Restriction sites Sequences prone to create secondary structures in RNA transcripts Effects on transcription (...

  10. 15 Hot Plasmids from 2017

    Type
    Blog Post
    ...repository, but be sure to let us know if you'd like to write about your plasmids in a future blog post. No...found at Addgene and in Figure S12 of the publication. Agudelo D, et al. Nature Methods. 2017. PubMed ...Rees H, et al. Nature Communications. 2017. PubMed PMID: 28585549 Kim Y, et al. Nature Biotechnology. ...GABA-ergic interneurons here. Dimidschstein J, et al. Nature Neuroscience. 2016. PubMed PMID: 27798629 Listen...Find PhoCl plasmids at Addgene. Zhang W, et al. Nature Methods. 2017. PubMed PMID: 28288123 Listen to...used as tools to learn about the influence of structure on an FP’s properties. pBAD-LSSmCherry1 is a long... Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169  pJL-SaBE3  SaCas9 (NNGRRT)    85170...

  11. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    ...CRY2PHR-activator fusion floats freely, depicted in the figure below. Once triggered by blue light, the CRY2 and...31X. By using a slit pattern during blue light exposure (470nm), the researchers showed that expression...created a photoactive Cas9 tool, as shown in the figure below. Their most successful design utilized Magnet...on and off. As one might expect from the modular nature of Cas9, Nihongaki et al. showed that it was possible...reach deeper tissues. Additionally, high intensity exposure can cause DNA damage. Far-red light, however, ...to create photoswitchable spCas9, dCas9, and S. aureus Cas9 (saCas9) proteins, showing the versatility... variant of AcrIIA4 (CASANOVA). As shown in the figure below, in the absence of light, Cas9 is bound by...

  12. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    ...guest bloggers Shravanti Surest from Iowa State University. Shravanti Suresh is a PhD candidate who investigates...This post was contributed by Shravanti Suresh from Iowa State University. Since its appearance, SARS-...19 and some serological tests to determine past exposure. However, the limited availability of reagents... turn to other technologies like CRISPR. Figure 1: An overview of the general schematic of CRISPR...with streptavidin, the lateral flow strip first captures the streptavidin portion of the probe via biotin...probe that is cut will continue to flow and be captured by an antibody further along on the strip. Highlights...Isothermal amplification is done at one constant temperature, eliminating the need for thermocyclers. This...

  13. Antibodies 101: Beyond Surface Labeling

    Type
    Blog Post
    ...a separate fix/perm cytosolic procedure in that case.     Figure 3: For intracellular labeling...antibody through the biotin-streptavidin interaction (Figure 1a). This interaction brings the fluorophore in...plasmids and biotin plasmids at Addgene!     Figure 1: Indirect detection of target markers can be ...(“anti-rabbit”) region of the primary antibody (Figure 1b). As above, the advantage of this system is ...which would label everything but the CD8+ T cells (Figure 2). Even better, we can use the same fluorophore... gating on the negative population.     Figure 2: Dump gating labels all unwanted cell lineages...membrane but inside the cell. Due to their chemical nature, antibodies cannot penetrate the cell membrane;...

  14. Antibodies 101: Introduction to Immunofluorescence

    Type
    Blog Post
    ...primary is conjugated to a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence...confocal   Figure 2: High-level overview of an indirect immunofluorescence procedure with key considerations...within a cell,” “Does this disease change the architecture of my cells,” or “How does this mutation impact...non-fluorescent visualization methods. Stay tuned for a future blog post to learn more about these techniques!.... For this strategy to work, it is important to ensure that you are using secondaries with fluorophores...divided into the several steps outlined below and in Figure 2. For a deeper dive into the steps and techniques...involve multiple smaller steps that all help to ensure that antibodies can access their target and that...

  15. Savvy Advocates Needed to Navigate a Scientific Enterprise in Flux

    Type
    Blog Post
    ...Symposium and Workshop, organized by Future of Research, Academics for the Future of Science, and the MIT Graduate... More information can be found at http://futureofresearch.org/advocating-for-science-boston-2016/. Sign...Science Symposium Boston 2016 Changes since the Future of Research Symposia Ongoing challenges facing...et al, PNAS 2014; Taylor, eLife, 2016). Several Future of Research (FoR) symposia held across the USA ...science to date. Advocacy opportunities for the future No matter your opinion on these changes, it is ...institutional responses to the FLSA ruling. Similarly, from purely a salary perspective, while this is a move in the...For those advocating in this area, keeping the pressure on for further increases will be challenging, ...

  16. Hot Plasmids: Spring 2025

    Type
    Blog Post
    ...Glypican Collection here! Figure 3: General glypican domain structure. Glypicans are proteoglycans ...— no UTR sequence added to the genome (Figure 4). Figure 4: STITCHR consists of a nicking Cas9 ...the FOS gene promoter and the MYC locus (Figure 5). Figure 5: TurboCas protein labeling compared ...synapses and deep-brain fiber photometry.  Figure 1: Characterization of iGluSnFR4 in mice. A) Scheme...choice. After hearing about researchers' favorite features and limitations of existing backbones, we're excited...viral generation and stable cell line creation (Figure 2). Looking for more? Explore our empty and EGFP...Addgene's own Lentiviral backbone plasmids here! Figure 2: Addgene cloned EGFP into pAG Lenti CMV N-HA ...

  17. Scientific Reproducibility - Focusing on Solutions at the Minisymposium on Reproducibility

    Type
    Blog Post
    ... notes on how lab procedures were actually performed, potentially curtailing future confusion (and hours...community-editable fluorescent protein database ReFigure: Save Scientific Figures into Dashboards and Share Your Insights...reproducibility issues in the biological sciences and featured speakers from academia, industry, nonprofits, ...reproducible if its findings can be predictably applied to future work. Importantly, no one should expect that every...Failed attempts to apply irreproducible results to future work cost both time and money with estimates reaching...doing an experiment wrong. However, even simple procedures like pouring a plate or running a gel have intricacies...initiatives to create more reliable reagents for future research. I’ve talked to many researchers who have...

  18. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ...factors to consider when choosing one.     Figure 1: Example of an experimental optogenetic setup...activity or gene expression. Experimental output is measured and quantified. Created with BioRender.com. ...either excite or inhibit neuronal activity upon exposure to light within a specific range of wavelengths... be used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light...engineered receptor that change shape with light exposure to activate them. PORTLs use a similar concept...when a signaling pathway is unknown.      Figure 3: Chimeric OptoXR incorporating rhodopsin undergoes...that may go awry in a disease state.      Figure 4: Example of an intracellular signaling pathway...

  19. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    ...10 minutes, but check with your enzyme’s manufacturer to ensure that you’re cutting for the proper duration... the ligation mixture with backbone alone; 2. Transformation containing ligation mixture with insert and...proper orientation. You then add ligase to the mixture to covalently link the backbone and insert and,... phosphatase) are commonly used. Follow the manufacturer’s instructions.   2. Isolate Your Insert and...unfortunately you can’t just throw the digestion mixtures together. You need to isolate your insert and ...charged DNA through the gel. As indicated in the figure on the left, your digested DNA (and undigested ... the Bitesize Bio Blog and their associated manufacturers websites. These stains require you to either...

  20. Chromoproteins: Colorful Proteins For Molecular Biology Experiments

    Type
    Blog Post
    ...which attract fish for them to prey upon (Figure 1). Figure 1: The sea anemone's red color can be used...aeBlue, amilCP, tsPurple, and gfasPurple) (Figure 2). Figure 2: Chromoprotein expression from bacteria...fluoresce at very low levels. Chromoproteins in nature Compared to the history of fluorescent proteins... chromoproteins lost color overnight in liquid culture, though expressing the chromoproteins from the ...chromoprotein plasmid that combines the most desirable features: intense color, fast maturation, and low fitness...     Chromoproteins for allelic exchange Figure 3: pTOX plasmids expressing amilCP and tsPurple...genetic modification integrates into the genome (Figure 3). Here, the chromoprotein is expressed and gives...

  21. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

    Type
    Blog Post
    ... active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components. Image from...it integrates into the trnS-leu2 locus.   Figure 2: Introducing Mobile-CRISPRi into bacteria. Top...However, the rate of transfer was variable. They measured transfer rates by comparing the number of colonies...several bacteria associated with human disease (S. aureus, Klebsiella pneumoniae, and P. aeruginosa) in addition...isolated from cheese. Knockdown efficiency, as measured by knockdown of a constitutely expressed RFP reporter...median knockdown of ~40-fold across all species measured. Beyond understanding the basic biology of bacteria...pathogens) were grown in presence of trimethoprim, cultures treated with a folA gRNA were more sensitive to...

  22. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    ...compositions of Cascade. Although seemingly ubiquitous in nature, they are largely underutilized in research settings...mostly a consequence of Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR types. Class 1 includes... systems utilize with type I-F Cascades. Figure 2: Cascade complex from CRISPR type I-E. Created...tools has been challenging. It can be difficult to ensure comparable expression of all components of Cascade...making cuts every so often. Cutting both strands ensures that there isn’t a template strand left behind ...behind that the cell can use to repair. Figure 3: General overview of the Cascade-Cas3 mechanism. Created...components can make Cas9 easier to work with than Cas3. Future targets Cas3 has the potential to be incredibly...

  23. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    ...MagLOV’s structure changes under magnetic field. Such changes could be harnessed or enhanced for future MagLOV-based...control of biological systems (Wheeler et al., 2016; Duret et al., 2019). But the field has sometimes seen ...1%) and recovered after the magnet was removed (Figure 1). At first, Andrew and Maria were skeptical. ... since the initial report in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in ...change of ~75% in response to the magnetic field (Figure 2); they named this variant MagLOV. Andrew was ...and didn’t need any added cofactors.      Figure 2: Fluorescence intensity and ΔF/F of MagLOV, a...magnet is waved under the sample. The imaged area measures approximately 82 mm across. Video courtesy of ...

  24. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    ...Martin, B., & Shendure, J. (2022). Precise genomic deletions using paired prime editing. Nature Biotechnology...editing world. The approach, originally published in Nature, is a “search-and-replace” genome editing technique... breaks (DSBs) or donor DNA templates.  Figure 1: An overview of prime editing. Created with BioRender.com...Liu lab contributed an improved prime editing “architecture” that can be combined with any of the PE2-PE5...prime editing as efficient as possible. Figure 2: Decision tree for selecting a PE6 variant. PE...2023, under a CC BY 4.0 license. epegRNAs: Structured RNAs with improved stability Traditional sgRNAs...of large DNA sequences with twin prime editing. Nature Biotechnology, 40(5), 731–740. https://doi.org/...

  25. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

    Type
    Blog Post
    ...green cards and citizenship.   Lauren Celano, CEO Propel Careers. Lauren is passionate about working with...This post was contributed by guest bloggers Lauren Celano of Propel Careers and Rachel Casseus, Esq. ...and on. As you think about your career (and your future in general), keep in mind that you may have years...careers - being aware of this can help take the pressure off having to find the perfect job immediately...but not limited to biotechnology, chemistry, architecture, engineering, mathematics, physical sciences...bachelor's degree or its equivalent and state licensure, if required to practice in that field. H-1B work-authorization...background, expertise, publications, and plan for future activities; including the interest of the greater...

  26. Getting to Know Expansion Microscopy

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    Blog Post
    ... 1) follows this structure: Step 1: fix (and maybe label) your sample (i.e. cultured cells or tissues...and after expansion and measure the degree of agreement between them to ensure the expanded images are...how you interpret a target’s structure. For example, cytoskeletal structures that form continuous lines ...easier, more accessible way to image biological features previously only visible via techniques like super-resolution...by just adding water.)  Step 5: Image   Figure 1: The ExM process allows you to physically magnify...depend upon the specific gel recipe used, ambient temperatures, incubation times, and the physical properties...properties of the sample itself. For example, cultured cells are typically easier to expand than mouse brain...

  27. Sharing Your Lab Protocols: Using Apps to Save Time & Track Your Experiments

    Type
    Blog Post
    ...Template). Nature Protocol Exchange Website: http://www.nature.com/protocolexchange/ Nature Protocol Exchange...for iOS and Android). A neat feature of protocols.io is the checklist feature which allows you to check ...process so you’ll have a record of your work. This feature further simplifies lab work if you like to bring...an open online protocol repository hosted by the Nature Publishing Group. Like the previously mentioned...and create lists of your favorite protocols for future reference. The exchange currently boasts over 2,500...links to protocols on their own websites. We're not sure how frequently it's being updated now, but there...access to protcols from external vendors, such as Nature Protcols and NEB. Quartzy Website: https://www....

  28. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    ...survive high temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C)....elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the...Reaction Temperature Calculator can be used to estimate an appropriate incubation temperature when using...will also efficiently ligate many undesirable structures, including substrates containing gaps or DNA ...phosphorylated base can be observed in the crystal structures of several DNA ligases bound to substrate (23... the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of annealed.... 2. Tomkinson, Alan E., et al. "DNA ligases: structure, reaction mechanism, and function." Chemical reviews...

  29. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

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    Blog Post
    ....  After this, lowering the temperature allows the repressor to re-nature and again repress expression...red recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are ...recombineering with an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering ...a generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering ... pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...few strains can also be found at Addgene, but be sure to read the associated papers for their particular...

  30. Plasmids 101: Origin of Replication

    Type
    Blog Post
    ...copies per cell. Figure 1: A plasmid map showing the standard features of a plasmid. So, how...Growth conditions The amount of aeration, temperature, culture volume, antibiotic, and medium can all affect...chloramphenicol – make sure your growth conditions aren't working against you!  The culture inoculum Freshly...colony and do not subculture directly from glycerol stocks, agar stabs, or liquid cultures. Freshly transformed...pairs can be separated more readily at lower temperatures and allows the replication machinery room to...if you need two plasmids for an experiment, make sure they have "compatible" ori's. See the table below... affect copy number. Some ori's are temperature sensitive; others ori's can be "tricked" into amplifying...
Showing: 211 - 240 of 1025 results