... 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...

...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...

...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...

... 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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...