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...3’ end binds template DNA for PCR amplification, while modifications and homologous sequences are encoded...multiple modifications A key feature of IVA cloning is the ability to combine multiple modifications. Any ...’ Check your amplification by gel electrophoresis. In our experience, good amplification after PCR means...gain traction? It is likely that both vector amplification with low fidelity polymerases and the cost of...this approach requires no special reagents or purification kits, it is low cost and can be immediately ...to produce the linear fragments, as desired modifications and homologous regions can be encoded in the...are perfectly fine, yet performing multiple modifications will require ultracompetent (109 CFU/μg) cells...

...far, we’ve used the MUCICAT system in two ways: In our initial paper, we used MUCICAT to optimize the gene...industrial strain. Not only is MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed...integration using CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these CRISPR systems...providing users with a complete editing process. The MUCICAT system While the above systems are great for experiments...chromosomal integration (ex: genome editing), our MUCICAT system which is based on the CRISPR-associated ...unstable and lack precise control over copy number. MUCICAT system contains 4 plasmids: pDonor, pTnsABC, pQCascade... crRNA can be customized as well, including concatenations into crRNA arrays targeting multiple different...

...from other sources. Mode of communication Different modes of communication take up different amounts of...your audience needs to understand what you’re communicating. While I’m sure that there are loads of ways...new languages!) Jargon has a place in science communication, but the more you use, the higher cognitive ...This can be obvious, in that a lot of science communication is helping people understand new concepts. You...intuitive, or logical to a layperson. When you’re communicating ideas that you think are clearly connected, ...pick and choose what concepts and details to communicate. It’s okay (and encouraged) to leave blank spaces...over some aspects when needed. The trick is to communicate your key points well enough that the audience...

...This application of AHA labeling is known as fluorescence noncanonical amino acid tagging (FUNCAT, Dieterich...experiments. Visualizing newly synthesized proteins with FUNCAT Professor Schuman’s technique applies metabolic...the whole de novo synthesized proteome. However, FUNCAT alone cannot be used to determine the turnover ...specific protein. Professor Schuman’s lab showed that FUNCAT’s usefulness can be extended to determine the turnover...ligation assay (PLA). The clever combination of FUNCAT and PLA PLA was originally published by the Söderberg...DNA strands take part in rolling circle DNA amplification. The amplified DNA segment is labeled with fluorescent...fluorescent probes that enable visualization of the location of the protein-protein interaction. To study protein...

...peptide (SpyTag) and protein (SpyCatcher) SnoopTag/SnoopCatcher SpyTag/SpyCatcher Rapid covalent bond formation...small, fast split intein SpyTag003/SpyCatcher003 SpyTag/SpyCatcher Rapid covalent bond formation between...SnoopTag) and protein (SnoopCatcher); no cross-reaction with SpyTag/SpyCatcher, allows for iterative expansion... expansion DogTag/DogCatcher SpyTag/SpyCatcher Rapid covalent bond formation between a genetically encoded...protein (DogCatcher); ideal for protein loop regions CRISPaint Gene Tagging Kit SpyTag/SpyCatcher CRISPR-assisted...and work well the first time. But biology is complicated — so many different parts and species, with components...integration of large DNA fragments at user-defined locations NEB Cell-Imaging Collection SNAP-tag, CLIP-tag...

...Description Origin of Replication (ORI) DNA sequence which allows initiation of replication within a plasmid...point for PCR amplification or sequencing. Primers can be exploited for sequence verification of plasmids...important. Our “Plasmids 101” series designed to educate all levels of scientists and plasmid lovers - serves...plasmids are small circular pieces of DNA that replicate independently from the host's chromosomal DNA....virulence. All natural plasmids contain an origin of replication (which controls the host range and copy number...Minimally, lab-created plasmids have an origin of replication, selection marker, and cloning site. The ease... plasmids and the ability of plasmids to self-replicate within a cell make them attractive tools for the...

...of complex post-translational modifications. Extraction and purification of recombinant proteins from ...modified via an array of post-translational modifications including protein folding, formation of disulfide...mRNA and the addition of post-translational modifications Learn more molecular biology in our Plasmids... experimental, biotechnological, and medical applications. Researches can visualize proteins in vivo by...expression systems used for protein production and purification. These include mammalian, insect, bacterial,...separated from contaminants using a variety of purification methods. Deciding which expression system to...protein you need   Your plans for downstream applications In this blog post we will summarize some of...

...results.  Replicates Replicates are another critical control in biological experiments. Replicates can be...of biological replicates such as multiple mice in each group and C, technical replicates or repeated measurements...secondary antibodies.   Application-specific controls Some antibody-based applications have very specific ...then moving on to controls for antibody-related applications. After reading, you will have a better understanding...blot and look for the molecular weight shifts indicative of tau phosphorylation. By including the positive...statistical power of an experiment. Technical replicates are repeated measurements of the same sample ...difficult to obtain meaningful results.  Biological replicates are distinct samples that are treated identically...

...Science Communication Conversation in Science Communication The Purpose of Science Communication #1 Mistake...supporters of science communication. To help you think about ways to effectively communicate your science, we...News at Harvard University. Effective science communication is required to promote public support for research... organization that works to promote science communication. Science in the News hosts a podcast, seminars...That’s right, #ActualLivingScientists, eager to communicate their science. In this first half of our two-...programs SITN runs and learn how they think about communicating science. Listen to the interview with Science...Mistake Scientists Make when Communicating with Non-Scientists How to Approach Different Audiences Listen...

... information you are looking for in our catalog or educational resources: plasmid, pooled library, blog...articles. From paper to plasmid Authors regularly indicate that their plasmids are available from Addgene...papers. Maybe they started their deposit prior to publication and were able to include specific plasmid ID ...terms we can use to find materials from this publication. Searching for the title of a paper works You...filters Not every plasmid search will start from a publication. You may have an idea for a plasmid that would...try different terms you may find the available categories and filters change as we now only show options... a second filter, this one from the ‘Requests’ category, will leave us with a single plasmid to consider...

...development, certification, degrees, foundations, coaching and even Twitter Chats dedicated to the Grant...the candidate application process, the candidate’s ability to write a grant application through a written... much more than writing. In fact, many grant applications and grant makers have moved away from long written...Now they require grant seekers to use online applications with significant character limits for the written...written portion of the grant application. They have increased their demand for stronger budgets with clear...duties can range from coordinating the grant application and submitting it, to evaluating the capacity...asked to serve on review panels and to review education materials for publishers. When the community orchestra...

...about which reagents were used? Unambiguous identification of a reagent is crucial for reproducibility...can lead to wasted time or retractions. Using a catalog number for identifying a reagent can be helpful...reagent online. Furthermore, if the link between the catalog number and reagent description is lost (e.g. a ... supplier is bought out by another company and catalog number changes) the original identifier loses its... but this approach has been adapted for the identification of physical materials that we use in our labs... which serve as unique PIDs within the Addgene catalog. These IDs allow you to unmistakably identify a...item. Given a specific Addgene ID # listed in a publication, you can quickly pull up the associated plasmid...

... post on mammalian vectors, will catch you up on the core replication and resistance features of yeast...recombination. Yeast Replicating plasmids (YRp):  These vectors contain an Autonomously Replicating Sequence (ARS...vectors must contain a yeast-specific origin of replication (ORI) and a means of selection in yeast cells...antibiotic selection markers.  The yeast origin of replication (Please note: This first section primarily pertains...ve also noted some features required for the replication of fission yeast, Schizosaccharomyces pombe, ...within a yeast vector determine how the plasmid is replicated and maintained within the yeast cell. These elements...integrated into the host DNA or is independently replicated as an episome. The four main types of yeast plasmids...

...sister locations on April 22, 2017, scientists and non-scientists alike will march to advocate for science...already come to pass. It may only be by advocating for science education and literacy that this changes.  2...marching for. I will be marching to advocate for science education and literacy as I believe people that...communities, microbial interactions, and science education.  Disclaimer: The views presented in this article...science’s place in education, government, and civilization in general (1). Science and politics? Science...process begins with a testable hypothesis - an educated guess about how some part of the world functions...health Immigration Pollution Healthcare Science education To make scientists relatable (17, 20). This...

...sensors One of the latest additions to Addgene’s catalog is a new class of sensors for neuropeptides and...doi: 10.1038/s41592-022-01597-x. New glutamate indicator iGluSnFR3 Improving on the glutamate sensor iGluSnFR...They generated variants optimized for different applications (v82 has higher affinity for glutamate but slower...PDGFR.codonopt    Aggarwal A, et al. Glutamate indicators with improved activation kinetics and localization...have developed a new genetically-encoded voltage indicator (GEVI) called JEDI-2P (Liu et al. 2022) optimized...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell. 2022 ...has generated a new genetically encoded calcium indicator using small monomeric NIR fluorescent proteins...

... you with two measurements: forward scatter (FSC) and side scatter (SSC). To acquire these measurements...exclude all duplicate events (two cells stuck together) from your single cells, as duplicates can’t be reliably...types of scattering that occur when a cell is pulsed by the laser. Within each type of scattering, you also...to keep it short though, promise! FSC and SSC: scattering To digitally extract your cells based on their...passing cell with a laser and detects the light scattered by that cell at a low angle (FSC) or large angle...: The laser pulse illuminates the cell and is scattered forward at a low angle (FSC) or to the side at...passes through the laser beam and causes it to scatter (FSC/SSC), the detected photons can be represented...

...Few Complicated Non-natural amino acids 1 Various Many Complicated   Applications Organic...new green fluorescent protein, some affinity purification tags, another version with that same red fluorescent... an enzyme that has been mutated to block its catalytic activity and instead become trapped in a covalent...having to clone a whole new construct for every application. Plus, the photophysical properties of chemical...advantages over fluorescent proteins, opening up new applications.   Figure 1: Live-cell labeling strategies...Although these multi-component systems can be complicated to implement, they can be highly specific with...self-labeling tag and a fluorescent protein) enables applications like multi-color imaging (Figure 2). If you’...

...production and purification that yield high titers (see also AAV protocols for production, purification and titration... are replication incompetent and cannot readily be passed on to subsequent cells via replication like ...intracerebral application, which is more invasive and has a much higher risk of complications. References...between different brain regions. For example, communication between the hippocampus and the prefrontal cortex...intracellularly spread away from the site of application and aid in visualization of the morphology of...flow in order to understand which brain regions communicate with each other and how they do it (i.e. where...where the signals come from and what implications their signaling is likely to have). Anterograde tracing...

...estimated scatterplot smoothing. (C) Number of requests per year for pooled libraries, categorized by function...insertion via a chemical modifier. We used these categories to see what kinds of CRISPR tools people were... pooled libraries, although we used different categories (Figure 2C). Note that in Figure 2, the numbers...Cumulative number of deposited CRISPR items, categorized by CRISPR function, by year. (B) Number of requests... years.  Libraries are divided into different categories based on function: knockout (KO), activate, inhibit...Figure 3, which shows requested CRISPR plasmids categorized by “age” or year they were deposited, you can...Percentage of requests per year for CRISPR plasmids categorized by year of deposit. Outlines show a representative...

...Restriction Enzymes + - PCR Amplification + + Digestion + + Gel Purification + - Ligation + - Transformation...vector is located in the 3’ end of the multi-cloning site, and the reverse primer is located in the 5’...Molecular cloning is the isolation, insertion and amplification of a recombinant DNA without sequence alteration... multi-step process consisting of digestion, purification, ligation, and transformation. While restriction...sequences for base pair overhangs, insert and vector purification, and, most importantly, excessive time requirements...is ligation independent, it does not require purification of insert or vector products, nor does it require...digested mixture. There is no need for further purification before transformation. After transforming cells...