We narrowed to 1,042 results for: URE

...lives of people and society. Future generations of IMP Motivating future generations is key to the success...internationalization become a reality available to all youth, our future, around the world.   In many countries of the world...improvement) and bright students that will be the future of the country. Brain drain is a serious problem...build, together with the universities, a bright future. IMP provides matched one-on-one international ...support to increase the chances of success, but also nurture a supportive and protective environment where individuals...empowered to productively navigate through both their failures and their successes. Although all mentorship relationships...their immediate frontiers. Moreover, sharing a culture/country of origin with an international mentor ...

...expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into kynurenic acid. That...That’s key because, unlike kynurenine, kynurenic acid can’t cross the blood-brain barrier. By lowering...muscles led to another clue: differences in the kynurenine pathway of tryptophan degradation. It turns out...lowering circulating levels of kynurenine in the body, the brain is protected from stress-induced biochemical...brain’s physiological demands,” they write. “In the future, these may lead to treatments for a variety of ...References: Skeletal muscle PGC-1α1 modulates kynurenine metabolism and mediates resilience to stress-...

...pellet for the remainder of the cultures and later maxiprep only the cultures with intact ITRs. I use a spreadsheet...proper packaging of AAV, but also form secondary structures that are prone to deletion from the transfer ...this issue. One way is to grow multiple bacterial cultures of your AAV transfer plasmid at 30 °C instead ...like to miniprep 5 mL of my AAV transfer plasmid cultures and then screen with SmaI digests. I freeze the...PEI transfections. It saves me time and helps me figure out if I have enough plasmid to complete my experiment... viral particles for efficient gene transfer. I figure it’s better to have a bit extra AAV than to have... skip the PEG precipitation of the HEK293 cell culture supernatant because it’s time consuming to prepare...

...synthetases.     Figure 2: Specific binding of scFVs to cognate aaRS antigens as measured by suspension ...encapsulating the protein of interest (Figure 3).   Figure 3: Schematic representation of the MPyV...domain (RBD) of the viral spike protein.   Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and...resolved fluorescence, and suspension bead assay (Figure 2). After finding no cross-reactivity, antibody...within seconds. A CRISPR-based lineage tracing tool captures rare clones that represent 0.001% of the population...found a way to use the accumulation of indels to measure time. These plasmids with different promoters driving...anti-CRISPRs were identified that inhibit the S. aureus Cas9, a Cas protein that is smaller and easier ...

...enabling microscopes to visualize the cellular architecture of whole tissues in 3D with unprecedented detail...thin sheet to illuminate a fluorescent sample and captured the reflected light using a different objective...Ultramicroscope was developed using a stereomicroscope to capture large field-of-views (6). For more comprehensive... on several other LSFM configurations’ various features see (4,7).   Light sheet fluorescence microscopy...methods will greatly aid our understanding of the structure-function relationship of the central nervous system...Jährling, C.P. Mauch,  K. Deininger, et al. (2007). Nature Methods, 4, 331–336. Pubmed. P. Keller, M.B. Ahrens...Neuron, 85, 462-483. Pubmed. E.H.K. Stelzer. (2015). Nature Methods, 12, 23–26. Pubmed. M. Weber, M. Mickoleit...

...your experiments. Capture MS Strategies This approach uses immunoprecipitation to capture proteins from a...been planning, it is a good idea to pause and make sure that your antibody has been appropriately validated...recognition of the target antigen. You want to make sure that your antibody binds to what you expect it to...application and available resources.     Figure 1: Table summarizing the IWGAV five proposed antibody...information for some antibodies, which makes it hard to ensure that you have unique antibodies. An additional ...and then uses mass spectrometry to identify the captured proteins. If the antibody is specific, then you...approach is ideal for validating antibodies for immunocapture applications and is amenable to high-throughput...

...design (meaning the mature processed form of crRNA is fused chimerically to a mature tracrRNA), the spacer... is based on the Cas9 D10A nickase described in Figure 4 of the Cong et al. (2013) Science paper. For ... to select your genomic target, you need to make sure the NGG immediately follows your target on the genome...-allelic cells usually make up the majority in culture unless the targeting efficiency is very high. Q6...NGA', or 'NGC' (if it's within coding region make sure it's a silent mutation). Q8: Is a protospacer ...the Cas9. Again, if it's within coding region make sure it's a silent mutation. Q9: When attempting to use...Restriction Fragment Length Polymorphism (RFLP) (see Figure 4 of the Cong et. al (2013) Science paper). If ...

...correspond to publications of popular papers. Figure 1: Plasmid requests by date. Parentheses depict...observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation of the effect of COVID...rough "COVID-19 effect" on order data, it does not capture the fact that different parts of the world encountered...='31668202421', style='text-align: right;' %} Figure 3. An animated map showing the plasmid orders and...during weekends and significant holidays. Figure 4: Plasmid request trajectories suggest an exponential...data likelihood with multivariate Gauss-Hermite quadrature, since the analytical expression for the data...winter break, and COVID-19 on plasmid requests Figure 5: Inferred effects. Values are the inferred percentage...

... the fluorescence amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have...rate. A rate of 2C/s is recommended to ensure an even temperature change among all of the droplets, but...what if we could repeat this assay 10,000 times to ensure that our titers were as accurate as possible? This...digital PCR involves partitioning a PCR reaction mixture into approximately 20,000 droplets using water-...containing thousands of droplets) can be sufficient to capture the information needed for your PCR experiment....droplet oil that you use should come from the same manufacturer. Otherwise you will end up with poor droplet...reader extracts the droplets from the plate and measures the fluorescence amplitude of each one. Droplets...

...plate to start liquid cultures. Lastly, you can start small overnight liquid cultures with the clones you...use depends on your preferences. Either way, make sure to test your colony PCR primers before using them...will need to screen.   Saving clones for later culture: At this point, you will want to hang onto your...bacteria-water suspension and use them to start cultures of your positive clones. If you want to store ...ones. Regardless of which method you choose, make sure to use the appropriate antibiotic for selection.... on an agarose gel to determine their size. Make sure to use an appropriate molecular weight standard ...samples before pipetting them onto the gel. The figure above summarizes generalized expected results for...

... Figure 1: Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from Zetsche...is also much simpler in structure than Cas9’s; only a short stem-loop structure in the direct repeat region...RNAs important for Cpf1 function, they found that mature crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides.... With this information, they turned to a cell culture system to see if any Cpf1 nucleases would exhibit... its proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively...deletions at the 3’ end of the target sequence. Figure 2: Effects of (A) double and (B) single base pair...Mismatch positions are indicated below each graph. Figure from Kleinstiver et al., 2016. Cpf1’s strength...

... way to grow haywire, access and create more vasculature to feed themselves, use the blood stream to commute..., introduced the “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized...the host immune mechanisms altogether and escape future immune surveillance. Dormancy of certain cancers... system to the presence of cancer are unable to mature into effector cells in the tumor microenvironment... look at a variety of up and coming therapies. Future directions and open questions in cancer treatment...constantly in flux and is dependent on the selective pressures placed upon it by its environment and the body...immune privilege and predicts reduced survival." Nature medicine 10.9 (2004): 942-949. PubMed PMID: 15322536...

...identify a few of the features that confer broad host range in plasmids in nature, and provide tips on ... identified some of the features that confer broad host range. These features are: The plasmid origin...characteristics of natural plasmids and how they function in nature can inform us on how to create better recombinant...on how we can harness those features to create broad host range recombinant vectors.  A plasmid’s host...are versatile and adaptable. Variation in ori structure allows plasmids to utilize diverse host replication...and reduced metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid...multiple oris that vary in DnaA-box and iteron structure, host-independent   Building broad host ...

...is to ensure the propagation of themselves and their associated genes. These systems better ensure that...that plasmids are passed down through generations (Figure 1). These so called “plasmid addiction systems”...the mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct... repeats. In cells where recombination has not occured, methanol induces the production of the toxic MazF...the mok SD resulting in a double stranded RNA structure that is recognized and cleaved by RNaseIII resulting...bioluminescence reporter in vivo in the absence of selective pressure and found that plasmids were maintained at 100%...with plasmid addiction systems While selfish in nature, these systems have been adapted by researchers...

...sites targeted by specific sgRNAs. Its RNA-guided nature makes CRISPRa much simpler to implement in genome-wide...transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct). More importantly...AAV or by mating with Cre-expressing mice. Figure 1: (A) Schematic of the Cre-dependent SPH construct...probe gene-function relationships in defined substructures of the CNS as well as individual neuronal populations...neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can be found here. Neuron-optimized...) with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church...including ubiquitous ones such as EF-1α and CAG. Figure 2: Dual lentivirus system with ubiquitous expression...

... 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...

... quality figure, produce the figure, its legend, and write a results section for that figure. Writing ...next manuscript 1. Be consistent with your figures The figures of a manuscript are the result of a lot of... to my PI about becoming a better writer, I was unsure if I should possibly take a composition class in... individual sections of a paper, the abstract, figures, introduction, etc. so as to thoroughly explain...quilt with your lab mates, you add the individual figures and results to make the final product with an interesting...Scientific Manuscript Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing Write for the...

...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...

...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...

... 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 ...

...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...

... 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...

...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...

...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 ...

...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...

...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...

...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...

...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 ...

...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...

...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...

...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...

... 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...

...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 ...

...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...

...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 ...

...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...

... 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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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!  ...

... 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...

...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...

...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...

...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...

...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...