We narrowed to 888 results for: URE

... of being a “good scientific citizen” and making sure you do what you can to help your fellow researchers...subtle, and it can take a lot of detective work to figure them out. But, when they come to light, it’s clear...way so other researchers can use them, to making sure that methods and protocols are available and detailed...Press we developed STAR Methods to improve the structure and transparency of methods sections, including... room to do more. If we could really instill a culture of rewarding good scientific citizenship in all...making it happen would increase dramatically. I’m not sure what the best approach to doing that will be, and...

...and cytoplasmic (CKB1) RNA target. Adapted from Figure 3 Colognori et al 2022.   Find CRISPR Csm plasmids...lagged. Fixation, chemical staining, and light exposure can negatively affect FPs during these processes... also withstands denaturing conditions, high temperatures, and other chaotropic conditions better than...these preparations. After crystalizing the hfYFP structure, the group rationally designed GFP variants with...fluorescence in high molarity GdnHCl, at high temperatures, and in the presence of oxidizing agents. Adapted...Adapted from Fig. 2 Campbell et al, Nature Methods   Find YFP plasmids here!   Campbell, B.C., et ...

...cleave them leaving mature crRNAs bound to the Cas9/tracrRNA complex. D) The mature crRNA is used to guide...GAA, or GAT SpCas9-NG 3' NG  Staphylococcus aureus (SA); SaCas9 3' NNGRRT or NNGRR(N)  Acidaminococcus...the crRNA to Cas9 and facilitates processing of mature crRNAs from pre-crRNAs derived from CRISPR arrays...unique stretches of DNA between them “spacers” (see figure below). After years of research, we now know that...sequences required for processing pre-crRNA into mature crRNA and tracrRNA binding. The spacer regions,...

...phosphorylation, promoting transcription (see figure below). Subsequent exposure to red light rapidly deactivates ...limited studies and can fluctuate with changing culture conditions. The use of low intensity light solves...promoting transcription of a GFP reporter. Subsequent exposure to red light rapidly deactivates the system. Version...systems The large dynamic range of these systems ensures that they can be used with many types of proteins...Scouras A, Ellington AD, Marcotte EM, Voigt CA. Nature. 2005 Nov 24;438(7067):441-2. PubMed. Find the...

...understanding the brain’s function is knowing its architecture, in particular the connections between different... uptake of these dyes in vivo, microinjection, pressure injection or iontophoresis (applying small electric...of RABV. This improvement resulted in a clearer picture of neuronal connectivity in the mammalian brain...but overall with rather low efficiency. Not all measures of transport efficiency are consistent which may...observations like these may be a starting point for future developments in the field of AAV vectors. Conclusion...achieve transgene delivery by targeting peripheral structures (such as injection into muscle) as opposed to...(2008). Concepts in the pathogenesis of rabies. Future Virol 3(5), 481–490. PubMed PMID: 19578477. PubMed...

...scientists and nonscientists alike). My only in-depth exposure to neuroscience prior to this conference was a.... This was not at all the case. The conference featured people who work in sociology, behavior, and artificial... session organized by Story Collider and which featured a talk about the different parts of the brain ...getting into neuroscience from the mol bio side you’re sure to have many opportunities to learn about and develop...website and on the Addgene blog to get some ideas for future experiments and to find tools that will make your...Ed Boyden on Optogenetics, Neuroscience, and the Future of Neuroengineering Primer on Optogenetics Resources...

...negative (digital) signal at the end of the reaction (Figure 1). Partitions that did not contain any target ...precision. This means digital PCR provides an absolute measure of target abundance, does not require standard ...directly at the predicted double strand break site (Figure 2A). The single molecule resolution of digital...completely “blind” to this very important difference (Figure 3). If you are interested in harnessing the power...requires target DNA sequencing to confirm the exact nature of your mutations. A great description of the plethora...induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology 31, 822–826 (2013). PubMed PMID: ...

...powerful approach for proteomic mapping in both cell culture and whole organisms (see our 2019 overview). Until...doi.org/10.7554/eLife.64631  Goertsen D, et al. Nature Neuroscience. 2022. https://doi.org/10.1038/s41593... applies both positive and negative selective pressures for novel capsids. In a continuation of that work...Find M-CREATE plasmids here!  Goertsen D, et al. Nature Neuroscience. 2022. https://doi.org/10.1038/s41593... factors, including limitations of number of flourescent proteins that can be inserted into a cell without...visual barcode plasmids here! Kaufman, et al. 2022. Nature Comm.https://doi.org/10.1038/s41467-022-30008-0...

...focus on antibodies that recognize proteins. Figure 1: Steps in the generation of hybridomas for monoclonal...fused to myeloma cells at a 1:1 ratio and then cultured in a special medium called HAT. This medium selects...antibodies are particularly useful when you want to ensure that the antibody binds to a specific region of... during your experiment, then you’d want to make sure that you choose an antibody that recognizes an epitope...polyclonal antibodies - which are a heterogenous mixture of antibodies that recognize many different epitopes...plasmid to create a recombinant antibody.     Figure 2: Changes to the heavy chain or light chain genes...

...densities are captured at the 17%, 25%, and 60% layers, and genome-containing virions are captured at the 40%...pun intended.)  Steps to clear writing Let’s use Figure 1 as our example. Remember, our goal is to use ...same information as sighted readers.    Figure 1: An image from AAV Purification by Iodixanol ...What are they hoping to get from it? The image in Figure 1 appears on our protocol page for AAV Purification...group to share with the rest of the workshop.  For Figure 1, Addgene chose this alt text: "The left panel...

...cell line cultures. Lentivirus Lentivirus is a type of retrovirus and shares many common features with gamma-retrovirus... gamma-retrovirus including genome architecture, host integration, and its association with disease. The...cell types. Altogether, AAV has many excellent features for human gene therapy and is currently being ...types of interest. This is a particularly nice feature as organ-specific injections, or the location of...   Table 1: Types of viruses and their features Choosing a virus Now that we’ve reviewed the...

...provided by the manufacturers and enables customized experiments. Projects such as the OpenFlexure Microscope...modify the device for individual experiments. Manufacturers rarely provide the construction plans or CAD...accessible open-source hardware. Projects such as the OpenFlexure microscope or open-source modular optical toolbox...documentation, which facilitates the reproduction of the structure in a similar way to the biological protocol, and...et. al, "Robotic microscopy for everyone: the OpenFlexure microscope," Biomed. Opt. Express 11, 2447-2460...

...important considerations for the future of open science? What about the future of open science are you most...do you hope Addgene will accomplish in the near future? Katz: I’m excited that Addgene is continuing to...those “what could go wrong scenarios” and making sure we have adequate process and controls in place to... particular area or profession, you need to make sure you have learned and mastered the fundamentals.   ...was a founding member of Mass Medical Angels, a venture mentor at MIT and served on the Boards of BioBuilder...

...and alternatives to fluorescent proteins. Figure 1: Firefly, from Wikimedia Commons. The North...reaction in its abdomen to produce yellow-green light (Figure 2). Despite the title of this post, this reaction...photon; no chemical reaction takes place. Figure 2: Simplified chemical reaction catalyzed by luciferase...luciferase gene, and expression of luciferase is measured via the light produced. A similar approach can...luciferase or alternative substrates can permit measurements of luminescence from live cells. Luciferase ...with experiments that supply both substrates and measure luciferase output at different wavelengths. Addgene...

...sequence, making them easy to harvest from cell culture media. Their His tags allow for further purification...for your RANBody While all three reporters stain cultured cells and tissue sections, some reporters are ...secondary antibody. Table 1: Summary of the Key Features of HRP, Spaghetti Monster, and Chicken IgY Domain...Tyramide signal amplification of HRP Staining cultured cells and tissue sections Amplifying weak signals...versions Labeled anti-HA or -Myc antibody Staining cultured cells and tissue sections When detected with a...(Y) Labeled Anti-Chicken IgY antibody Staining cultured cells and tissue sections Great for multicolor...

...and work quickly to mitigate RNA degradation. Make sure to use DEPC-treated or RNAse free water. If using...water, autoclave the water to inactivate the DEPC.  Ensure that plasticware or glassware used is RNase free...or more large, expensive ultracentrifuge.  Figure 1: An outline of the different steps in RNA extraction...you need really clean RNA, it’s important to make sure that when extracting, to only take the aqueous layer... from) on your resuspended RNA sample will help ensure DNA contamination is not an issue.  References...thiocyanate–phenol–chloroform extraction: twenty-something years on. Nature Protocols 1:581–585. https://doi.org/10.1038/nprot...

...types. Be sure to check the spectral properties of the fluorophores you are considering to make sure that ... peptides, like an Fc-binding domain (Figure 1).  Figure 1: Examples of sites on antibodies used...process quick and easy. Before getting started, be sure to check that your antibody’s buffer is compatible...2017) Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14:865–868. https...

... is limited and many microbes are difficult to culture outside of their natural environment. Thus, the...Pamela Silver’s lab developed a synthetic mark and recapture strategy for bacteria to count cell divisions....generations can be determined. The lab used DCDC to measure growth rate of E. coli for over 10 generations ...reveals growth dynamics in the gut microbiota." Nature communications 6 (2015): 10039. PubMed PMID: 26615910...engineering of the mammalian gut microbiome in situ." Nature methods 16.2 (2019): 167. PubMed PMID: 30643213... analyzing gut microbiota and host biomarkers." Nature communications 9.1 (2018): 3347. PubMed PMID: 30131493...

...understood, a team of researchers lead by Takayuki Kogure (Kogure et al., 2011) believes that miRNA may decrease...metastasis." Nature 527.7578 (2015): 329. PubMed PMID: 26524530. PubMed Central PMCID: PMC4788391. Kogure, Takayuki...Microenvironments are cellular regions with specialized structures. Tumors are surrounded by microenvironments and...instead of repressing them, making it even harder to cure cancer. To understand how cancer cells influence...

...the biggest project I’d seen in my already long tenure at Addgene. I would work with our research team...of what we discussed and decided. I needed to a) figure out the sources of miscommunication and b) find... steps towards solving this problem by 1) making sure we qualified these words when we discussed them ...Creating this guide was relatively easy. I took a picture of virtually every container we used, then for ...team identify possible points of confusion in the future. Improving cross team communication, be it in ...productive. Higher Productivity. Taking active measures in improving cross team communication has helped...

... are to figure out the best way to shepherd technologies through thorny ethical issues, ensure that everyone...communities, which is in turn crucial if we want to make sure that everyone can be informed and empowered to make...public engagement efforts, including giving public lectures, writing science articles for a lay readership...organizations (often volunteer-based) that put on public lectures, science cafés or science festivals, and getting...events will be important both in preparing you for future work in these areas, and in helping you decide ...

...a large undertaking. Priority number one was to ensure that this changeover had no impact on our ability... Since we’ve successfully grown the plasmid in culture to prepare it for sequencing, we feel confident...proteins, and selectable markers by detecting common features using Snapgene. If we find information that differs...many of you will be familiar with our efforts to ensure access to high quality, ready-to-use viral preps...Sequencing (CPS) workflow and yield sufficient data to capture the entire viral genome. The full details of that...it on to our new MiSeq and set up the run making sure to note the appropriate barcode for each sample....

...tools to tease out the details underlying brain structure and function, with the advent of each tool expanding...excitatory and inhibitory synapses in the brain.  Figure 1:Schematic diagram of different FingR constructs...protein products have distinct localizations. Figure 2: Split-wrmScarlet labeling proteins with different...more red than blue (Eastman et al., 2020). Figure 3: BaF3 cells were transduced with H2B-FT virus...which depend on IL-3 to proliferate. The cells cultured with a low dose of IL-3 showed reduced proliferation...proliferation and had a greater number of red cells as measured by flow cytometry. Cells grown in high IL-3 showed...

...process. Industry interviews, however, were more structured. I met with several individual and/or small groups...a theme too, which is something not inherently captured by the STAR method. I like to think of the theme...evaluations, professional development, and company culture.  It’s also a good idea to prepare 1-2 unique questions... of your work, such as improving a process or procedure, solving a problem, or making a key decision. ...hours, which was a lot of extraversion for me. Make sure to get enough sleep the night before. Have your ... professional  First impressions matter, so make sure yours counts. Dress professionally but still like...

... without DNA cleavage. Nature 551:464–471 . https://doi.org/10.1038/nature24644 Gaudelli NM, Lam DK, Rees... editors (ABE) mediate A•T-to-G•C base changes (Figure 1), but it can be challenging to make these base...and dCas to the genomic DNA of interest  Figure 1. When ABEs are in action they bind DNA so that...NG-Cas9 (PAM: NG) to create NG-ABE8.xm/d and the S. aureus Cas9 (PAM: NNGRRT) to create Sa-ABE8.x-m/d. With...increased activity and therapeutic application. Nature Biotechnology. https://doi.org/10.1038/s41587-020...

...You’ve analyzed the data and generated beautiful figures. And, you can’t wait to tell your story. But, before...accomplished with an exciting title and attractive figures. Next, the poster should be legible from a distance...central box, often used for displaying all of one’s figures, or at least the majority of the visual data. This...poster sections like the introduction, methods, and figures flank the central region, and a scannable barcode...potentially more catchy, but it can be trickier to make figures fit nicely into circular shapes rather than a square...you select for the background, you’ll want to make sure that the color of your font is compatible. Use high-contrast...

... the lynch pin that results in your success or failure and demands attention, practice, and honing of ...otherwise why would you be interested in it?) so make sure that comes across. Check out some in-depth grant...you were awarded money to carry out research, be sure to make it clear what the outcome of the work was... the successful use of your preliminary data to secure further funding. Make it clear that you intend ...productive with the granting agency’s investment. 6. Failure is part of the process, keep your head up! It’...an IDP Get Tips on Keeping Up With Scientific Literature  ...

... multiple gRNAs on the same plasmid, you’ll make sure that each cell that gets the plasmid contains all...blog post about multiplexing with Cpf1.   Figure 1: Multiplexing allows researchers to express multiple... illustrated in the schematic below.   Figure 2: gRNA target sequences (colored rectangles) are... Note: depending on which method you use, the procedure will vary slightly.   Two Golden Gate options...need to supply it with another plasmid.   Figure 3: Comparison of Multiplex Strategies including...chromosomal rearrangements with the CRISPR/Cas9 system.” Nature 516(7531) (2014): 423-7. PubMed PMID: 25337876....nucleases for highly specific genome editing.”  Nature Biotechnology 32(6) (2014): 569-576. PubMed PMID...

...into polypeptide chains that make up the primary structure of proteins. Most proteins are then modified via...localization or purify proteins to study their structure, interactions and functions. Proteins can also...easily synthesized, must be produced using complex mixtures derived from cells or using live cells. There ... expressions systems do require demanding cell culture conditions compared to other systems. Insect expression...consuming and that insect cells require demanding culture conditions similar to mammalian expression systems...expression. Plant systems share many of the same features and processing requirements as mammalian cell ...

...a formal training for new managers and I had a “Eureka” moment while reviewing the results of my Myers-Briggs...easily identify what to delegate and to whom (make sure that person has time for it!). Then schedule a meeting... I still need to check myself sometimes to make sure I am delegating well, but it does get easier over...able to go to your manager for advice if you’re not sure how to face a particular situation. But keep in ...reason!).  Try to buffer them from unnecessary pressure “coming from above”, which is when other skills.... Three Rules for Managing a Successful Team. Naturejobs blog by Joanne Kamens  Are Good Managers Born...

...intermediate to advanced and finally, to expert.   Figure 1. Visual representation of common injection routes... injections require a little bit of practice to ensure the proper injection site has been located and ...instillation” or “gavage,” as they do not require any punctures. Intratracheal instillation Intratracheal instillation...delivery is also common. Due to the intubation, this procedure is considered quite invasive. Restraint: anesthesia...similar to its intratracheal counterpart, as both procedures target the lungs. The main differences are that...on the end, which prevents accidental tears or punctures to the throat. The feeding tube can be flexible...

...University School of Medicine. It’s not always easy to figure out what you want to do after graduate school, ...experience taught me something about myself and my future career. If it wasn’t something I liked – well, ...the labs in our program to write short, funny, picture-filled blog posts about their research? Then we.... I gained a concrete example to use to promote future attempts and learned a bit about recruiting scientists...shape, was when I started volunteering. I wasn’t sure yet what a career in science outreach would look... disappointed graduate students because I wasn’t sure what I wanted to do. A few weeks into volunteering...

...as part of President Barack Obama's Winning the Future Initiative. Endy's team made news in March with...plasmid kits, the Biobricks Public Agreement, and the future of the bioeconomy. Addgene: What do these BIOFAB...life, then this new language needs to be free. The future of synthetic biology Addgene: You've described ...visualization. Addgene: What do you envision for the future as this programming language is put into practice...therapeutic space. If you were to look forward to the future of the bioeconomy it's less clear, but the way ...standard transcription and translation initiation." Nature Methods. 10, 354-360 (10 March 2013). ...

...approximately 1,900 sgRNAs and identified sequence features and adjustments that improved sgRNA activity and...libraries (see below). Let’s take a look at the features and advantages of the optimized human CRISPR libraries...scientists to conduct genome-wide screens in standard cultured cells as well as more difficult conditions such...sgRNAs for CRISPR-Cas9–mediated gene inactivation." Nature biotechnology 32.12 (2014): 1262. PubMed PMID: ...and minimize off-target effects of CRISPR-Cas9." Nature biotechnology 34.2 (2016): 184. PubMed PMID: 26780180...activation by an engineered CRISPR-Cas9 complex." Nature517.7536 (2015): 583. PubMed PMID: 25494202. PubMed ...

...issues raised by team members, and plan for the future of the team. All while still paying bills and having...These skills will be useful anywhere I work in the future; they are transferrable across most, if not all...you’re sitting in the lobby of (hopefully) your future employer. Your heart is pounding, you’re short ...communication style, your potential teammates want to be assured that you will contribute openness, clarity, approachability...your boss needs someone to step up, when a new venture presents itself even outside of work, consider ... these skills you’ve used in your own work - I’m sure you’ve used many! Teamwork Collaborating with others...

...providing many of the details not generally captured in the literature, scientists can use the Data Hub to compare... process is often not detailed in the primary literature, and as a result can leave scientists under-informed...both excellent at infecting our target neurons (Figure 1). However, this one set of experiments didn’t...indicated they wanted to see more negative data (Figure 2.) At the same time, when scientists who support...Initiative, F. B. (2019). Setting your data free. Nature, 5–7. O’Connor, D. M., Lutomski, C., Jarrold, M...

...rescue known pathogenic alleles, or introduce a premature stop codon to render an RNA nonfunctional. In ..., like ADAR2DD(E488Q). They also adjusted the structure of the guide RNA by introducing a C opposite the...delivery.  Find the RESCUE RNA editing plasmids here! Future directions Building on previous RNA targeting work...Omar O., et al. “RNA targeting with CRISPR-Cas13.” Nature 550(7675) (2017):280-284. PMID: 28976959. Find...enable guide-RNA processing and RNA detection." Nature 538(7624) (2016):270-273. PubMed PMID: 27669025...Resources on the Addgene Blog Check out our CRISPR Featured Topic Page Learn about RNA Targeting with Cas13a...

...Chromatin folds into secondary structures (loops) and tertiary structures (globules) to achieve exquisite... enhancers with the help of genome math. Figure 1: Genome math illustrated. Intersect (left) and...which genes influence the survival of our cell culture is the foundation of modern drug discovery and ...periodically use next-generation sequencing to measure the relative proportion of guide RNAs (gRNAs) across...for further details. DNA is a 3D Fractal Figure 2: DNA compaction. Graphics courtesy of beyondthedish...

...From the beginning, one of Addgene’s goals was to ensure the scientific community obtained high-quality ... programs to a uniform language.  The modern architecture of Nextflow included built-in support for containers...convenient compatibility with our internal software infrastructure; however, it is not necessary to use containers...input channel to only use the first n items. To be sure, we went through much trial and error to learn the...with our bbduk process.    At first, to make sure we understood the concept of channels and implemented...compared to purchasing and maintaining our own infrastructure. Our patchwork collection of scripts evolved...

... without DNA cleavage. Nature 551:464–471 . https://doi.org/10.1038/nature24644 Glaser A, McColl B, Vadolas...APOBEC-Cas9n-UGI complex. So how exactly does this work? Figure 1: APOBEC- and Cas9-mediated editing (ACE) reporter...mCherry positive cells (Martin et al., 2019). Figure 2: Editing efficiencies using base editing reporters...visualized and sorted via flow cytometry. Figure 3: Targeting pEF-BFP with a cytidine deaminase ...detectable protein, not just fluorescent proteins. Figure 4: Reporters in Gene On include a mScarlet reporter...