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We narrowed to 1,025 results for: URE

Showing: 301 - 350 of 1025 results

  1. The Open Repository of CRISPR Screens: CRISPR Screen Data in One Place

    Type
    Blog Post
    ... identify. Figure 2: Users can cross-interrogate screens from the literature for a drug, disease...particular screens of interest.   Figure 3: Each publication featured in BioGRID ORCS has a dedicated summary...large-scale datasets by computational biologists. Figure 1: The BioGRID Open Repository of CRISPR Screens...compiling genome-wide datasets, which are often obscured in large supplementary data files, BioGRID ORCS.... Importantly, metadata for each screen is captured by BioGRID curators, including the cell line/tissue...determine the gene hits for each screen are also captured as metadata.   Screen results are reported as ...comprehensive pre-compiled downloads and via a full-featured REST Web Service API. Both options are fully open...

  2. Plasmids 101: TOPO Cloning

    Type
    Blog Post
    ... of these methods to ensure your insert retains the 3' A overhang: Use a mixture of proofreading enzyme...restriction enzymes or ligase, and requires no post-PCR procedures. Sounds easy right? The technique relies on the...how does TOPO cloning work? As illustrated in the Figure below, the “A” overhang on the blue PCR product...insert, the magic of TOPO cloning happens. Basic procedure Let’s break down the steps needed for TOPO cloning... TOPO Vector.  3. Incubate 5 Minutes at Room Temperature: You can place your reaction on ice if you are...extension time after the last cycle of PCR to make sure that the "A" gets added to all PCR products. Keep...electroporation to fail). When incubating at room temperature, it is not recommended that you exceed the 5 ...

  3. Antibodies 101: Epitope Availability

    Type
    Blog Post
    ...assays, such as ELISAs and Westerns, frequently denature the proteins as part of the sample prep. This ...it from its native state to a less folded, or denatured, one. This enables absorbing of the proteins to... or ELISAs may therefore often be specific to denatured versions, or even to just that particular assay...    Fig. 1: A native protein (left) is denatured, allowing the antibody to bind to a previously...you’re working with native proteins instead of denatured ones - well, don’t relax just yet. Proteins often... state or specific configuration. So how do you ensure that you have the right antibody for the protein...Did you have a denaturing step? Do you want to capture the protein bound to anything? Is it a transmembrane...

  4. Three Tips for Preventing Viral Plasmid Recombination in Your Samples

    Type
    Blog Post
    ...marker and origin of replication. During bacterial culture, only the plasmid with the antibiotic resistance... that contain the full length plasmid.   Figure 1: The full lentiviral plasmid is expected to be...researchers find that using a 30°C incubation temperature to grow bacteria more slowly helps reduce the...Esposito (2018) noticed that in Stbl3 bacterial cultures with lentiviral vectors, the flat white colonies...tan colonies that look similar to normal E. coli cultures contain the recombined vector backbone. Test multiple...multiple colonies before saving a glycerol stock to ensure the colony you selected contains the full plasmid...length plasmid. If you find that your bacterial cultures contain a mix of full length plasmid and the recombined...

  5. CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

    Type
    Blog Post
    ...mushroom escapes US regulation. Nature 532:293–293 . https://doi.org/10.1038/nature.2016.19754 Zhang H, Demirer...Fan C, Landry MP (2019) DNA nanostructures coordinate gene silencing in mature plants. Proc Natl Acad Sci...sufficient to modify the genome of plant cells. The structure of the gRNA (composed of a ~20 nucleotide targeting...components is essential for any CRISPR experiment, and failure to express either the gRNA or Cas9 in your cell...into a target cell line or organism (Presented in figure 1). More information on Agrobacterium-mediated ...mediated cloning of your gRNA of interest. Figure 1: Simplified schematic of agrobacterium-mediated... (carbon nanotubes, Kwak et al., 2019), DNA nanostructures (Zhang et al., 2019) or cell-penetrating peptides...

  6. 10 Steps to a Perfect Science Talk

    Type
    Blog Post
    ... not being interviewed, there could always be a future boss in the room, so it is a good idea to start...academic position, see this very good article at NatureJobs: Interviews: The All-Important Job Talk. 1. Watch...Scientists do this all the time, you just need to figure out how to make it suspenseful. 4. Start preparing... Google.slides or Powerpoint file right now to capture interesting key results, striking images, creative...Of course, if some of your work does relate, make sure to include that material, but don’t force it. You...explaining how your work fits in the bigger science picture. Scientists especially like to learn new things...resulted from their research, “aha” moments, and future research directions. Don’t worry if your work hasn...

  7. Addgene Depositors Get More Citations

    Type
    Blog Post
    ...our journal-adjusted citation measure we re-weight the 3 percent of Nature papers without Addgene plasmids... (=C*G) 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1...also pulled the big interdisciplinary journals: Nature, Science, PLOS One, and PNAS. We then broke the...papers without plasmids deposited with Addgene (Figure 1). Even aggregated this much, you can see the... Addgene plasmids in our data were published in Nature, but only 3 percent of papers without Addgene plasmids...papers with and without Addgene plasmids are in Nature. This is also true for other journals, so our estimates...Environmental Microbiology  1.6% 1.2%  0.75   125.3 In figure 2, you can see the full impact from using these...

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

    Type
    Blog Post
    ...spectrophotometer measures these absorbances using UV-transparent cuvettes. First you measure the absorbance...in. This is a “blank” and measures the background absorbance. Then you measure the absorbance of the DNA...DNA sample.  These absorbance measures give you an idea of the concentration of your DNA prep and whether...Generally, A260 of 1.0 is equivalent to 50 ug/ml pure dsDNA. Use the following formula to estimate your...as compared to spectrophotometric methods that measure all nucleic acids). These methods are more sensitive...quantified first before larger fragments. Figure 1: The quantification of a RNA ladder using capillary...sensitivity so it’s not used much. However, the measurements are made in the visible range and can be read...

  9. Celebrate National Book Month with Addgene

    Type
    Blog Post
    ...Austen’s Pride and Prejudice, and some more obscure treasures, like Tove Jansson’s The True Deceiver. There...on-site book swap/library, reading is part of the culture here at Addgene. Since it’s National Book Month...own hands. Who doesn’t love a book series that features a young female protagonist, some clever magic,... may be intimidating, Ruthefurd’s writing will capture the attention of even the most skeptical reader...club Indeed, reading is ingrained into Addgene’s culture. We have a book club in the office that was founded... had a few reasons for launching this one. On a purely selfish level, I wanted to be challenged to read...has become one of my favorite parts of Addgene’s culture. Have you read any of these books? Whether you ...

  10. Using CRISPR/Cas9 to Edit Disease Out of the Genome

    Type
    Blog Post
    ...absolutely possible to use CRISPR to cure genetic disease in the near future,” Li says, though there are some... derived from iPSCs. From Rohn et al. 2018; see figure 3 for full details.    CRISPR advances in monoallelic...receptor (CFTR) by homologous recombination in cultured intestinal stem cells of patients with cystic ...When all was said and done, the researchers had cured 24 mice of their disease. Meanwhile, Hans Clevers...because the genomes of adult stem cells in organoid cultures are stable,” said Gerald Schwank, first author...off-target effects is a major limitation. The procedure will also need to be perfected until the efficiency...of repair approaches 100 percent. “We need to be sure it's accurate enough and efficient enough for repair...

  11. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    ...strand in Figure 1 below) was free to anneal to complementary ssDNA molecules in vitro. Figure 1: Cas9...presumptive mechanism of action are presented in Figure 2. Figure 2: Single-stranded donor DNA interaction...Understanding Cas9 biophysics enhances HDR We first measured Cas9 dissociation rates and discovered that the... of approximately 6 hours. To our surprise, we measured identical dissociation rates for catalytically...catalytically inactive dCas9 produced a “bubble” structure in uncut target DNA that was accessible to anneal...single stranded DNA. We thus wondered if such a structure could drive sequence replacement in cells. Targeting...inactive CRISPR-Cas9 using asymmetric donor DNA." Nature biotechnology (2016). PubMed PMID: 26789497.  2...

  12. Management for Scientists: Delegating is Key

    Type
    Blog Post
    ...make sure the work gets done and done well. Involving your team effectively is now your new measure of ...Setting someone up for failure by delegating is a bad idea. How do I make sure it goes well? As with ...appropriate communication between members of the team to ensure that no component of the overall project gets left...fun, but getting buy-in when delegating can help ensure successful results.  Can you provide the information...will be made that could have been prevented.  Make sure everyone understands that no question is “dumb” ...and plan changes. Written documentation helps make sure everyone is on the same page. Effective delegation... the best use of your time and skills. It also ensures that other people in the team grow and develop ...

  13. How Addgene Creates Content

    Type
    Blog Post
    ...process led to quick dives back into the literature to make sure absolutely everything was correct. Final... we usually make figures in BioRender or use appropriately licensed and cited figures from papers or image...content - the useful information on scientific literature, technical skills, and experiences from thousands... Nearly everything we write entails a primary literature search to collect up to date information on the...Final touches We try to use figures and images to help illustrate our points and break up walls of text...graphic designer made most of the Antibody Guide figures. This requires a fair amount of communication between...our content team and our graphic designer to make sure things are clear, accurate, and in line with Addgene...

  14. A Practical Approach to Choosing the B(right)est Fluorescent Protein

    Type
    Blog Post
    ...brightness in a system closely mimicking the future application. In figure 2 we provide an example of the comparison...spent and, if you do it right, can be (part of) figure 1 of your next paper or thesis. This series of ...protein is based on the properties found in the literature or tabulated data that summarizes properties ... and therefore high brightness is an important feature of a fluorophore. The key question is how the brightness...detector sensitivity) and the biological system (temperature, prokaryote versus eukaryote, background fluorescence...within the dish. This is caused by the stochastic nature of plasmid uptake by cells. To correct for this...2010). An explanation of the assay is depicted in figure 1. The strict correlation between the fluorescent...

  15. Antibodies 101: ChIP

    Type
    Blog Post
    ...interactions. ChIP works by using antibodies to capture protein:DNA complexes with antibodies specific ...your protein(s) of interest. Once the complex is captured, it is treated so that the protein and DNA are...Once you have your sample, the first step is to ensure your protein:DNA complexes will remain bound to...size, typically between 200-1000 bp. This is to ensure that your downstream analysis focuses on the DNA...the sample, introduces bias due to the selective nature of the cutting. While sonication is usually the...DNA and then determine concentration.  Protein Capture After crosslinking and sonication or enzymatic ...native proteins (so antibodies validated only for denatured proteins may not be a good choice) and the amount...

  16. Advice for Starting a Biotech Company from a Grad Student turned Entrepreneur

    Type
    Blog Post
    ... unable to secure enough money to sustain a startup, he went to work for Flagship Ventures – a local Cambridge...in academia and industry vary drastically, he reassures that one can learn it by reading a broad variety...of materials and constantly practicing. “The expenditure of effort in preparation and practice is directly... Cambridge biotech venture capital firm with deep ties into the local academic community. However, Mike...entrepreneur must be OK with the constant fear of failure at all stages in the startup". To gain insight ...and vision for the company; this will help avoid future complications and potential conflicts that waste...takes around 1 – 2 years. The product is the measure of success. Technology is proven in (A) Productize...

  17. Viral Vectors 101: AAV Serotypes and Tissue Tropism

    Type
    Blog Post
    ...between strains of one species, so be sure to check the literature and validate your choice of AAV vector...adeno-associated virus infection. Nature, 530(7588), 108–112. https://doi.org/10.1038/nature16465 Rubin, J. D., Nguyen...allowing them to target particular cell types. Figure 1: Diagram of AAV components. Created with BioRender.com...vectors have both distinctive capsids and genomic structures. The viral capsid is the key that lets AAVs “...mixing, matching, and modifying capsid and genomic structures. For example, rAAV pseudotypes mix the genome... by Addgene are pseudotypes using the genomic structure of AAV2, so they are formally called rAAV2/1, ...and so on, although we often use abbreviated nomenclature (AAV1, AAV2, etc.) to distinguish the capsids...

  18. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    ...about are (1) your experimental conditions and measurements; (2) the cells you’re targeting; (3) how much...vector: serotype, promoter, and titer.     Figure 1: Flow chart depicting a) steps for selecting ...injection site than AAV2 (Watakabe et al., 2015) (Figure 1). You’ll need to consider if such properties ...goals when designing and testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and...regions.  Image modified from Aschauer et al., 2013, Figure 1. Used under Creative Commons Attribution License... 2004; Taschenberger et al., 2017).     Figure 3: Comparison of two astrocyte selective promoters...neurons (NeuN). Image from Griffin et al., 2019, Figure 4. Used under Creative Commons Attribution 4.0 ...

  19. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    ...to the system and in what order they were added. Figure 1D below demonstrates an example of a register ... in certain states of interest. For example, in Figure 2 below we present a two-input, five-state GRSM...we implement RSMs in E. coli with two plasmids (Figure 3 below): Input Plasmid: The input plasmid (high... RSM (See performance data from the indicated figures in our paper (1)) Input Plasmid Output Plasmid ...designed to have 20-30 nt length, ~60 oC annealing temperature, and ~50% GC content) so that individual parts...Ara) RSM over a time series of increasing input exposure lengths and found that two hour inductions were...each possible permuted substring of inputs (see Figure S9 of our paper (1)). We anticipate that, depending...

  20. Viral Vectors 101: An Introduction to AAV

    Type
    Blog Post
    ...systemically administered in an organism. These features make AAV ideal for human therapies and in vivo..., many of which are available in rAAVs!   Figure 1: Diagram of AAV components      Cargo capacity...gene switches, and/or serotype can also be used to ensure specificity; this is particularly helpful when ...differentiated, non-dividing cells.   Figure 2: DNA rescue and transduction of a conventional...uncoating in the target cell nucleus, this DNA structure can readily fold into transcriptionally active...double-stranded form through intra-molecular annealing. Figure and legend from Okada, 2013.   scAAV If your gene...As a starting point, we recommend checking the literature and our AAV data hub to see if others have used...

  21. Celebrating 15 Years of Scientific Sharing

    Type
    Blog Post
    ... achieved and the opportunities we have for the future. At Addgene we will never ever stop planning, perfecting...re meeting the challenges of DNA distribution to ensure scientists everywhere can access plasmids despite... team. We show up in both science journals like Nature and in mainstream media like The Atlantic. We’ve...working in lock step to maximize the services and features we can offer online for Addgenies and to serve...Addgenies strong. The Addgene Board of Directors is mature and poised to support our growth. Thanks to Joanna...thinking, and openness to team work.   Company culture Once in awhile I worry a very little bit about...Best Place to Work with a positive, supportive culture, we are attractive to the local employee pool and...

  22. Antibodies 101: Selecting the Right Antibody

    Type
    Blog Post
    ...well for applications that denature a protein to its primary (linear) structure using heat or acid, for ...Picture this: you’ve been assigned an exciting new project aimed at understanding how a critical cellular...the application-specific data provided on the manufacturer’s website. Because of how they are developed,...Take a look at the publications cited on the manufacturer’s website, which may contain more detailed data...in a single experiment then you will need to make sure that you can easily distinguish each different type...epitope target, source, and other information to make sure you’re not trying to choose between the same antibody... partners by mass spectrometry (Uhlen, 2016). Be sure that the antibody you choose has been validated ...

  23. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
    ...are doing multiple digestion be sure that the buffers and temperature are compatible between the different...or use a different temperature (30°C) for the growth on plates and in liquid culture. If you don’t get ...mindful of the restriction enzymes you chose. Make sure that enzymes that you are using are not blocked ... few general parameters that you can adjust: temperature and time of incubation, and amount of DNA. As...a few colonies (5-10) and grow them in a small culture volume (2 or 3 ml) containing the corresponding...following steps: Do a single or double digest to be sure that the plasmid is the correct expected size and...acknowledged in hundreds of publications including Cell, Nature, and other journals.   Paolo Colombi is currently...

  24. 5 Steps to Writing Grant Proposals for Grad Students

    Type
    Blog Post
    ...review the literature and offer educated insight into what the desirable features of future technologies...communication skills, Elucidates the big picture and future directions of the project, Outlines a detailed... proposed work. Thus, every single sentence and figure must be clear, concise, meaningful, and supportive...months to give feedback on the application. Make sure to invest time and consider the reviewer's comments...grant writing comes with continual practice and exposure, here are some additional links that offer more...first grant proposal" by Soapbox Science Editor in Nature Blogs "How to write a research project grant application...

  25. CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9

    Type
    Blog Post
    ...transcription, and splicing. Nature. 2016;539(7629):452. doi:10.1038/nature20149 Jiang Q, Meng X, Meng L, ...this may still unexpectedly affect nearby genes (Figure 1A-C). Find CRISPR plasmids at Addgene! Knock-in...will terminate transcription of most of the ncRNA (Figure 1D). While this method can be time-consuming, it...-target effects (Zibitt et al. 2021).    Figure 1: A schematic illustration of CRISPR/Cas9-based...insertion of a synthetic polyadenylation (spA) signal. Figure and caption adapted from Hazan and Bester 2021....nearby genes and interact with specific chromatin structures or other local genomic elements (Hazan and Bester...CRISPR/Cas9-targeting of long non-coding RNA genes. Nature biotechnology. 2020;38(5):573. doi:10.1038/s41587...

  26. Antibodies 101: Stripping and Reprobing Western Blots

    Type
    Blog Post
    ...evenly re-absorb aqueous buffer. Be sure to check the manufacturer’s instructions for the membrane you ...companies including ThermoFisher, Millipore Sigma, and Azure Biosystems offer pre-made stripping solutions or...solution and agitate for 5–30 minutes at room temperature. The incubation time will depend on your blot... stripping solution uses heat and detergent to denature and remove antibodies but is also likely to remove...also volatile and toxic (and smells bad!), so be sure to use it under a fume hood. Rinse the membrane...Assess the stripping Now it’s time to check to make sure the antibodies from your first round of detection...remains, you may want to repeat your stripping procedure, perhaps using longer incubation times, or try...

  27. Tips for Technical Support Calls

    Type
    Blog Post
    ...consider before you commit: Double check to make sure the answer to your question didn’t come with your...means their original articles are your best bet to figure out how to use a particular reagent.  We also have... improve your customer service experience: Make sure you’re contacting the correct company. This might...possible about the issue. For Addgene support, make sure to mention the plasmid catalog number, or at least...experts on every technique or experiment, but a picture of a gel or a sequencing trace file can go a long...long way in helping us understand the nature of your concern. Please be courteous. We understand how... support that I miss is that you’re never really sure what you’re going to get. There are questions that...

  28. Visualizing Your Plasmid Request Data

    Type
    Blog Post
    ...excited to see that Addgene has recently added a new feature for depositing labs to its website, allowing depositors...aggregated data. Plotting the data in interactive figures is a great step up, but I needed a way to show ...Addgene Plasmid Request Data Dashboard! Figure 1: The Addgene Requests Dashboard   The dashboard...following items (Fig. 1): A row with four of the key figures Number of requests per reagents (as a horizontal...request data to the dashboard (Fig. 2).     Figure 2: Insert showing the upload function for the Addgene...depositor and the date of downloading the CSV is shown (figure 2) and the dashboard displays the data in a standardized...organization(s) is not shown on the dashboard. Key features of dashboards are (i) easy access to important...

  29. Finding Your Science Policy Path

    Type
    Blog Post
    ...others (see more in Figure 2). Starting early demonstrates not only your interest to future positions, but ... Individuals that work in science policy are a treasure--they love the work and the mission so much that... Luckily, there are many mechanisms in place to ensure your effective networking---and they’re more accessible...of science with a large set of other societal pressures including economic, cultural, and political. Science...relations, advocacy, and writing legislation (see Figure 1 for career map). The beautiful part of science...more about science policy as a profession. Figure 1: Science policy can lead you to many paths! For...sabbatical year or transition away from academia as tenured professors. There’s never a bad time to start your...

  30. The Addgene Antibody Data Hub

    Type
    Blog Post
    ...the antibody performed.  Figure 5: Results for the data report shown in Figure 3. The antibody is rated... how an antibody performed in an experiment.   Figure 1: The front page of the Addgene Data Hub.   Antibody... whether the results include knockout data.    Figure 2: Search options in the Antibody Data Hub. Data...find that information in the report as well.    Figure 3: An antibody data report, showing the results...description of the experimental conditions.     Figure 4: A screenshot of the materials and methods section...section for the data report shown in Figure 3.   At the end of the report, you’ll find a results section... To submit, simply click "Contribute Data."    Figure 6: Contributing data to the Data Hub is easy!  ...

  31. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    ... cloning efficiency measurements. We performed additional efficiency measurements using similarly sized...assembly Single insert cloning efficiencies were measured based on the acquisition of antibiotic selectable...), while sequencing of white colonies showed a mixture of mis-assemblies and very occasional uncut or ...Golden Gate Assembly All Golden Gate assemblies feature an inverse proportionality between the complexity...containing the transformants onto the selection plate. Figure 2 shows representative transformation plates obtained...assemblies and over 90% for 24-fragment assemblies (Figure 3, Table 1). These improvements (5- to 12-fold ... per plate using outgrowth volumes described in Figure 3, with calculated yields from entire outgrowth...

  32. The Scientific Conference Poster Session: Tips for Success

    Type
    Blog Post
    ...papers or “hot topics,” so be sure to be well-read on the recent literature in your field. Take your time...about to present your first scientific poster, but unsure how best to prepare. Maybe you’re a presentation...attending, your preparation and day-of game plan will ensure you have the most beneficial scientific conference.... For example, when thinking about discussing a figure, jot down the question you were trying to answer... to say and provide your audience with a clear picture of your work. Practice, practice, practice: Rehearse.... Finding your poster board well ahead of time ensures you aren’t scrambling to tack up your poster seconds... the poster, as opposed to engaging with you? Be sure to use your poster as a visual aid, not a crutch...

  33. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    ... to learn more about nanobodies and how their structure and function compare to IgG antibodies, as well...llamas, alpacas and other camelids. In terms of structure, HCabs are like a pared down version of a standard... nanobody, HCab, and traditional IgG antibody structures. Functional comparison Strong but Large: Advantages...big enough that multiple labels (green circles in figure 2 below) can be attached to each antibody. But...standard IgG secondary antibody (Pleiner et al figures 4A-C). And since they have limited cross-reactivity...colocalization labeling of up to 3 targets (Pleiner et al figure 4D). Unlike large secondary antibodies, small nanobodies...eliminating the secondary antibody incubation. See figure 4A below for comparison of this one-step staining...

  34. DNA Purification Without a Kit

    Type
    Blog Post
    ...plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid DNA This kit-free plasmid miniprep...extraction kit. First, you lyse the bacteria and denature the DNA and proteins in solution. The pH is then...glass syringe filters Starting material: bacterial cultures or agarose gel slices Product: plasmids or DNA...hydrophobic glass filter. Each glass filter can capture up to 150 ug of DNA, and filters can be stacked...together in series to increase binding capacity. Figure 1: Purify DNA using glass filters. Image: Kim and...filters you use depending on the size of bacterial culture you’re working with. Overall, the glass syringe...and without degradation during the extraction procedure. Additionally, purified DNA was free of agarose...

  35. INTEGRATE: Bacterial Genome Engineering Using CRISPR-Transposons

    Type
    Blog Post
    ...the ability to easily cure cells of the plasmid after integration. Figure 2: (A) Streamlined VchINT...species not previously cultured or isolated (Rubin et al., 2020).  Figure 3: Multiplexed insertions...transposon end sequences (R and L ends).  Figure 1: (A) Components involved in each type of CRISPR-Transposon...produce off-target DNA insertions and intermediate structures known as cointegrates (Vo et al., 2020; Strecker...achieved with VchINT, at two different incubation temperatures. Data adapted from Vo et al., 2020. In E...targeting by a transposon-encoded CRISPR–Cas system. Nature 577:271–274 . https://doi.org/10.1038/s41586-019...–Cas systems direct RNA-guided DNA integration. Nature 571:219–225 . https://doi.org/10.1038/s41586-019...

  36. CRISPR 101: Ribonucleoprotein (RNP) Delivery

    Type
    Blog Post
    ...for delivery of RNPs is electroporation (A in the figure above), which generates pores in the cell membrane... addition to the use of this technique in cell culture, it has also been applied to genome editing of ..., such as lipid-mediated transfection (B in the figure above), are in the early stages of development....Cas9 proteins harboring receptor ligands (C in the figure above), which result in the internalization of ...transfection of protoplasts and biolistic bombardment of immature embryos (Liang et al. 2017).  PEG-mediated methods...equipment, such as a gene gun. Depending on the nature of the experiment, it is up to the researcher to...protein-based genome editing in vitro and in vivo." Nature biotechnology 33.1 (2015): 73. PubMed PMID: 25357182...

  37. Summer SciComm Series: Modes of Communication

    Type
    Blog Post
    ...science communications: podcasts, blogging, videos, lectures, exhibits, social media… how do you choose? Which...Them All, consider the following four questions to figure out which method(s) work best for you, your audience...-speed internet. Before you begin any work, make sure you know if your audience will be able to access...never know what's going to work. After all, a stick-figure internet comic about math probably didn't sound...needed. And since we live in the digital age, make sure to research any software you may need: cost or freeware...accessible or most appealing to you. And if you’re not sure what all the possibilities are, check out our table... useful for data-focused stories. In-person lectures, presentations, etc… Low (except for space) Real-time...

  38. CRISPR Antimicrobials

    Type
    Blog Post
    ...proportion of virulent S. aureus from 50% to 11% in just 24 hours.   Figure 1: Single-phage strategies...targeting sequences found only in certain bacteria (Figure 1). Citorik et al. targeted beta-lactam and quinolone...al. used a phagemid system to target virulent S. aureus, a common hospital-borne infection. Two rounds ...system to resensitize antibiotic-resistant bacteria (Figure 2). The first phage introduced is lysogenic, integrating...resistant bacteria have previously flourished.   Figure 2: Two-phage strategies to resensitize bacteria...CRISPR, we’re turning the tables on the bacteria. Future research will show if this new strategy can help...using efficiently-delivered RNA-guided nucleases.” Nature Biotechnol. 32(11): 1141-5. (2014). PubMed: PMID...

  39. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    ...infection with S. aureus (red). You can then see neutrophils swarming around S. aureus and eating the bacteria...largely absent in adult organisms, where tissues structures are more stable and local movements predominate...than the static microscopy images of chemically coloured lymphoid organs did in the past. How do researchers...electronic transition in a fluorescent molecule (see figure to the right). The excitation generated by these... a chosen focal volume and thus the microscope captures only fluorescence coming from this volume. The...environments. These studies provide quantitative measurements of cellular motility, interactions, and response...peripheral interfollicular region of lymph nodes." Nature immunology 9.2 (2008): 155-165. PubMed PMID: 18193049...

  40. How to Write a Scientific Review Article

    Type
    Blog Post
    ...about citing: I wasn’t sure if I should cite reviews or the primary literature or both. I ended up citing...lists and pictures are the most useful parts of reviews. These could be in the form of figures/schematics... to write, to read efficiently, to search the literature, and to synthesize a large volume of information...well-researched field, you may need to get specific to make sure your article doesn’t turn into a textbook. Get the...active/well-known researchers in the field. I made sure to find information from genome-wide studies, as...appreciate the discussion sections of the primary literature as a resource that both summarized results and...Addgene Blog Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing Tips for Improving...

  41. Plasmids 101: Gateway Cloning

    Type
    Blog Post
    ...recombination sites (attL-left- and attR-right-, Figure 1). Under certain conditions, the attL and attR... regeneration of attP and attB sites.   Figure 1: Lambda phage integration and excision reactions...gene is excised from the donor vector.   Figure 2: The Gateway system adopts phage integration ... program to design Gateway plasmids.    Figure 3: Method A to create an entry clone: recombination...into an attL-containing entry vector.   Figure 4: Method B to create an entry clone: TOPO cloning...) of an attL-containing entry vector.   Figure 5: Method C to create an entry clone: Restriction...clone to create the expression clone.    Figure 6: Generating an expression clone. The reaction...

  42. Changing Labor Laws Bring Increased Postdoc Wages

    Type
    Blog Post
    ...This post was contributed by Future of Research Executive Director, Gary McDowell. On Dec 1st, the threshold... guidance issued by the Department of Labor. At Future of Research, our volunteers have been helping to...their postdoctoral salary scales to the NRSA scale (Figure 21, National Postdoctoral Association Institutional...legal justification of this have not been received. Future actions for junior scientists and for academia ...many postdocs will immediately lose jobs; that tenured professors/mid-career scientists/junior faculty...biomedical enterprise in the U.S. will decrease. At Future of Research, we think this is a good thing. Reliance...McDowell is currently the Executive Director of Future of Research. He is interested in advocating for...

  43. pCXLE toolkit: Efficient episomal plasmid-based method to reprogram peripheral blood cells to iPSCs

    Type
    Blog Post
    ...requires stringent biosafety measures, thereby increasing the costs of the procedure. Episomal reprogramming...fibroblasts, which require a more invasive skin biopsy procedure. This becomes a major benefit when recruiting ...reprogramming success [1]. Fibroblasts need to be cultured and expanded in vitro for several passages to ...collection [2]. PBMCs display unique epigenetic signatures that are closer to hiPSCs than age-matched fibroblasts....03%) [3, 9]. In 2011, Chou et al. generated a mixture of two vectors that encoded six factors - OCT3/... system generated by the group is an optimized mixture of three episomal plasmids (Table 1) encoding the...cells with unique epigenetic and gene expression signatures. Cell Res, 2011. 21(3): p. 518-29. PubMed PMID...

  44. Advanced Uses of Cre-lox and Flp-FRT - A Neuroscientist’s View

    Type
    Blog Post
    ... at the nucleotide level but share an overall structure of 13 base pair palindromic repeats separated ...efficient The first version of FLP discovered has a temperature optimum of 30 °C and is therefore inefficient...led to the identification of FLPe which has a temperature optimum of 37 °C (Buchholz, Angrand, and Stewart...Visualizing single neurons in the living brain   Figure 1: Plasmid mix to label neuronal morphology (eYFP...transfection efficiency. However, if the cellular structure of a neuron and its synapse should be visualized...Combing FLP and Cre for additional control   Figure 2: Expression of a morphological marker (eYFP) ...FLP Recombinase Evolved by Cycling Mutagenesis.” Nature biotechnology 16(7): 657–62. PubMed PMID: 9661200...

  45. Binning Singletons: Tackling Conference Networking When You Don’t Know Anyone

    Type
    Blog Post
    ...minutes to point me in the right direction for my future work. She graciously took me for coffee and spent...conference for any reason was a Singleton. Figure 1: The Binning Singletons logo. Image: James et...that binning Singletons together would provide a structure and support network for people attending large...improvement. We will make a greater effort in the future to match people within similar fields (ASM Microbe...that all participants would benefit from more structure with regard to the Mentor-Singleton relationship...addressing. Clear expectations have been established for future participants regarding the activities and time ...scientific community can improve the diversity and culture of science. We want Binning Singletons to be another...

  46. Overcoming the AAV Size Limitation for CRISPR Delivery

    Type
    Blog Post
    ... Staphylococcus aureus Cas9. Nature 520:186–191 . https://doi.org/10.1038/nature14299  Senís E, Fatouros...bacteriophage and plasmid DNA. Nature 468:67–71 . https://doi.org/10.1038/nature09523  Ibraheim R, Song C-Q, ...SI, Dohmae N, Ishitani R, Zhang F, Nureki O (2014) Crystal Structure of Cas9 in Complex with Guide RNA ... pool of shorter orthologs, only Staphylococcus aureus Cas9 (SaCas9, 1053 amino acids) displayed cleavage...a distinct family of RNA-guided genome editors. Nature 566:218–223 . https://doi.org/10.1038/s41586-019...

  47. Important Considerations in Optogenetics Behavioral Experiments

    Type
    Blog Post
    ...automatically using motion capture software such as EthoVision. Consult the literature to find all the applications... is an approximation of reward or the sense of pleasure that the animal perceives through taking a drug...may affect the animal; stimulation could induce seizures or some other unwanted phenomenon or it could ... elegans [10]. If used properly, optogenetics is sure to teach us much in all of these species. Happy ... nucleus accumbens facilitates reward seeking." Nature 475.7356 (2011): 377-380. Pubmed PMID: 21716290...direct comparative analysis of microbial opsins." Nature methods 9.2 (2012): 159-172.5. Goshen I, et al...."An optogenetic toolbox designed for primates." Nature neuroscience 14.3 (2011): 387-397. PubMed PMID:...

  48. Who Gives a Tweet? 9 Facts About Scientists on Twitter

    Type
    Blog Post
    ...scientists was Nature. Overall scientists tweet URLs with scientific domains <10% of the time. Figure 3: Top ...by biologists were: Nature, Sciencemag, Wiley, Cell and Scientific American Figure 4: Top Scientific Domains...have the most Twitter users. Biologists rank #8. Figure 1: Highly Represented Scientific Disciplines on...scientists on Twitter are male and 40% are female Figure 2. Gender Ratio of Scientists on Twitter. 60% of...scientific domains more often than other scientists   Figure 5: Portion of Tweets with Scientific Domains, Categorized...their fields than scientists outside their fields Figure 6: Follower Network of Scientists on Twitter. Each...

  49. Starter Guide to induced Pluripotent Stem Cells (iPSCs) Part 2:  Reprogramming and Transdifferentiation

    Type
    Blog Post
    ...Jaenisch, Nuclear reprogramming and pluripotency. Nature, 2006. 441(7097): p. 1061-7. PubMed PMID: 16810240...cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663...human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc Natl Acad...differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res, 2009...pluripotent stem cells into intestinal tissue in vitro. Nature, 2011. 470(7332): p. 105-9. PubMed PMID: 21151107...Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons. Sci Rep, 2015. 5: ...Dedifferentiation, transdifferentiation, and reprogramming: future directions in regenerative medicine. Semin Reprod...

  50. Understanding GPCR Signaling Using TRUPATH

    Type
    Blog Post
    ...updated version of BRET called BRET2. Figure 1: TRUPATH measures heterotrimeric G protein dissociation...can be measured by a BRET2 ratio, which is the ratio of GFP2 emission to RLuc8 emission (see figure). What...? The TRUPATH collection allows researchers to measure the physical association of combinations of different... signaling pathways.  This dissociation can be measured using a technique known as Bioluminescence Resonance...Rluc8 was more challenging. The Roth lab used structure-guided protein engineering and experimental refinement...both antagonists and inverse-agonists could be measured using the TRUPATH system. Conclusions Although...
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