...coupled with heterotrimeric G proteins on the intracellular side of the cell membrane. Heterotrimeric G proteins...in signal transduction pathways and consist of a trimer of three subunits: alpha (Gα), beta (Gβ), and gamma...the GPCR allows for binding of a new inactive heterotrimeric G protein complex and subsequently another ...elucidate the role of 14 of the 16 non-visual heterotrimeric G proteins that can be activated downstream... alpha, 2 beta, and 4 gamma subunits of the heterotrimeric G protein complex that can be used to detect...

...strengthened during memory formation and memory retrieval. In neuronal tracing experiments, brain tissue...Traditional tracing compounds either produce a colorimetric product or are fluorescent. While dyes usually...pressure injection or iontophoresis (applying small electric currents via inserted electrodes) may be applied...both antero- and retrograde labelling, synaptic restriction as well as controlled monosynaptic and polysynaptic...polysynaptic spreading. AAV vectors cover some of these attributes and their use has allowed for the visualization...recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem 109, 838–845. PubMed PMID...PMID: 19727139. Ugolini, G., Kuypers, H.G., and Strick, P.L. (1989). Transneuronal transfer of herpes ...

...your cloning strategy: Restriction enzyme digestion Be mindful of the restriction enzymes you chose. Make...This post was contributed by Oskar Laur, head of the custom cloning core at Emory University, and Paolo...which technique you are going to adopt (i.e. restriction cloning, Gibson Assembly, Golden Gate etc.), ... fragments of the size you expect, check the restriction pattern of the enzymes you chose, and verify ...hopefully have some new strategies to tackle that tricky cloning project. Good luck with your experiment...

...translation by sequestering the RBS, but binding of the “trigger RNA” (in this case, biomarker RNA or 16S rRNA isolated...can also detect expression of toxin mRNA from Clostridium difficile. As mRNA expression cannot be identified...fluorescent markers to visualize localization and distribution of gut bacteria without fixation or preparation... count cell divisions. The technique, called distributed cell division counting (DCDC), relies on the ... for both in vitro and in vivo studies. Find distributed cell division counting plasmids here References...PMCID: PMC2752045. Myhrvold, Cameron, et al. "A distributed cell division counter reveals growth dynamics...

...March 2015, Addgene reached the 500,000 plasmids distributed milestone! We were thrilled to see the impact...one plasmid deposited at Addgene that has been distributed more than 100 times.   “I really like this award... labs worldwide use our plasmids. I hope they contribute to great discoveries!” — Julien Béthune, Professor...Flame Award on Bluesky. In 2016, we also began distributing ready-to-use viral vectors from some of our ...supportive educational resources. By 2017, we had distributed 1,000 ready-to-use viral vector preps. Today,...citations of individual items in our repository, a tribute to how many discoveries have been made because ...

... added level of confidence in the plasmids we distribute through our quality control processes. Every ... Addgene, we collect data on every plasmid we distribute through our deposit process, and then we carefully...functional mutations, antibiotic resistance, restriction sites used during cloning, and most importantly...sequencing reactions in parallel. We have a few tricks to simplify the process. First, nearly all of our...sequencing from the exact same stock that we store and distribute from. Of course, your culture should always be...sequencing reads? Tell us your favorite sequencing tricks in the comments section below.  Additional Resources...

...183 gene therapy trials (http://www.abedia.com/wiley/vectors.php). These clinical trials have been directed...This post was contributed by guest blogger Harshana S De Silva Feelixge. Gene therapy technologies ...gene therapies from conceptual design to clinical trials involve identifying a therapeutic gene, finding...most naturally occurring viruses used in clinical trials, wild type AAV is not associated with any pathogenicity...While basic research and other ongoing clinical trials support the utility of AAV as a broadly applicable...

... caps, compatible with your centrifugation speed, to cover the centrifuge bins. These have to be added...okay to add another level of protection! When centrifuging viruses (a necessary step during production)...significant risk for aerosol contamination. As most centrifugation has to be done outside of a biosafety cabinet... and removed in a biosafety cabinet. For ultracentrifugation, heat sealed tubes can be used for extra ... spinning down lentivirus, you’ll need to use centrifuge bin covers, which you should open in the biosafety...

...science has become increasingly restricted to academic and industrial labs, but recently there has been ...The following post was contributed by Derek Jacoby from Makerspace Victoria, CA Over the decades science...possible that we’ll find something interesting and contribute it back to the kittybiome project, but the primary...generally funded on a membership model - everyone contributes each month - with only occasional supplementation...would otherwise be discarded. For instance, the centrifuge in the picture had a broken controller and would...

...volume of lysate passed over the binding matrix could clog the matrix when performing filter based plasmid...DNA to verify your plasmid via sequencing or restriction digest. Chances are, you’ll even have DNA left...media You can achieve higher cell density using a nutrient-rich broth such as Terrific Broth, 2xYT, or a ...longer growth time. Note: Julian Taylor-Parker contributed to the writing of this post. Additional Resources...Transformation Learn How to Perform a Diagnostic Restriction Digest Additional Resources Sigma-Aldrich Introduction...

...commercial lateral flow strips, similar to pregnancy tests. With lateral flow strips, the presence of your...determined by the number of bands present on the strip. This type of readout allows for nucleic acid detection...detection almost anywhere as lateral flow strips are easy to transport and work rapidly, providing reliable...platform to detect SARS-CoV-2 using a lateral flow strip format. They published this in Nature Biotechnology... validation studies.     Jennifer Tsang contributed to updating this article. References Abudayyeh...

...subjected to quality control by Addgene before being distributed to customers. Details about our production protocols...control are described below. AAV Production AAV distributed by Addgene has been produced either in-house ...preparations are purified by iodixanol gradient ultracentrifugation and concentrated to purity and titers adequate...preparations is assayed by comparing the relative stoichiometric ratios of the viral capsid proteins VP1, VP2...containing lentivirus is first cleared by low-speed centrifugation and then by filtration through a 0.45 µm polyethersulfone...in 10% polyethylene glycol (PEG) followed by centrifugation. Precipitated pellets containing viral particles...

...Glove trick Putting on lab gloves can sometimes be annoying. To make it easier, try this quick trick. Just...Addgene has distributed over 800,000 plasmids since 2004. In that time, our lab has gotten a lot experience...Just flip, and press! Lab tip #2: Tube flick trick When combining reagents in the lab, it is important...several times. This will help ensure ensure equal distribution. Lab tip #3: Barcode everything Use barcodes...

...from 5,821 labs in 1,249 institutions across 55 countries.  Our first request was on September 8, 2004 (...pcDNA-p160MBP, if you're curious.) Since then, we've distributed 2,002,539 materials to 254,034 scientists in ...in 8,900 institutions across 111 countries. Just eight days into 2024, we've already shipped 2,078 items...scientists at 375 organizations in 21 different countries. It's safe to say we keep our shipping team busy...international community, we've partnered with distributors in China, Korea, Japan, India, and Brazil.  ...

...publication and we have already collected over 100 entries spanning six different species and dozens of different...tool hM4D(Gi) driven by a GFAP promoter in rat. Contribute your AAV data Just like Addgene’s plasmid repository.... We are grateful to the scientists who have contributed data so far and we encourage you to submit data...the data hub website as a thank you for your contribution to open science.  Please note that the AAV Data... currently only accepting data for materials distributed by Addgene.  Future directions As the AAV Data...

...This post was contributed by guest blogger Joachim Goedart, an assistant professor at the Section of ...genetically encoded fluorescent probe. Several factors contribute to the popularity of GFP including (i) fast and... mutation (A206K) is necessary to generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al... bind the molecule of interest. For instance, pleckstrin homology domains can be used to detect specific...have successfully generated several functional heterotrimeric G-alpha subunit fusions that do not tolerate...Merel JW, et al. "Real-time visualization of heterotrimeric G protein Gq activation in living cells." BMC...

...This post was contributed by guest blogger Joachim Goedart, an assistant professor at the Section of ...several ways to measure stickiness. In vitro, ultracentrifugation or gel filtration is the most common analytical...mScarlet-tubulin (Plasmid #85045). We often use restriction enzymes in the multiple cloning site for generating...Bendezú et al., 2015) and the alpha subunit of heterotrimeric G-proteins (Janetopoulos et al., 2001, Adjobo-Hermans...Merel JW, et al. "Real-time visualization of heterotrimeric G protein Gq activation in living cells." BMC...Devreotes. "Receptor-mediated activation of heterotrimeric G-proteins in living cells." Science 291.5512...

...screen using PCR and a restriction enzyme digest.   Fig. 3: Restriction enzyme screening for resistivity...of your experiment for a screening strategy. Restriction enzyme screening is sensitive to single base ...changes, but the edit must occur at an existing restriction enzyme recognition sequence or generate a new... but your edit doesn’t affect any endogenous restriction enzyme sites? Never fear! You can introduce a...silent mutation should be designed solely for restriction enzyme sensitivity or resistivity, and thus be...

...forward to in 2023 and beyond. Last year, we distributed 165,000 plasmids, including 27,000 CRISPR plasmids...and general-use antibodies, is available in both trial- and full-size, with assay-supported recommended...our website saw 2.2 million visitors from 231 countries. Guides and protocols were our most popular content... million page views from visitors in over 200 countries. We, like everyone around the world, are learning...research community through sharing science and distributing high-quality materials and information. As a...

...When performing restriction digests on plasmids at Addgene, we sometimes observe something odd in our...various multimeric forms (e.g. concatemers, dimers, trimers, tetramers). Since then, multimerization has been...themselves cannot be used to detect multimers, since restriction sites in multimers usually repeat in tandem along...DNA without changing the size. As a result, a restriction digest of multimers will produce the same fragment...sample, you could try linearizing the plasmid via restriction digestion and then run the linearized plasmid...eBookAddgene's Molecular Biology ReferenceAddgene's Restriction Digest Protocol More resources on the Addgene...

...This post was contributed by guest bloggers and IMP organizers Rosario F. Godino, Rocío López-Diego, ...youth, our future, around the world.   In many countries of the world, professional internationalization.... Brain drain is a serious problem in several countries, however programs like IMP, bring closer than ...from talented professionals to their original countries to help build, together with the universities,...pre-program self-assesment. Achievements directly attributable to participation in the program included improvements... will also initiate mentoring pilots in other countries. In 2016, we expect the program to comprise over...

...nights, game nights, and trips to eat out together. “We also have a lab day trip each year and as we do ... the lab receives send-off via a gift, card, and trip to the pub. Dr. Ellis adds “we probably don’t celebrate...lot of yeast research, a few of those have been trips to breweries who happily exchange our expertise ...company outside of the lab by sharing meals, going on trips, and going to New Hampshire in the summer for the...

...through a distributor?  Yes! Packaged on Request service will be available through our distributor partners...of AAV plasmids will have technical or legal restrictions that make them ineligible for use in this service...international; varies widely by country and/or distributor.)  You should plan to receive your Packaged on...is launched, you’ll be able to work with the distributor to place a request and order if approved.  I ...

...on chemical (CaCl2, cationic liposome) or electrical (electric current forming membrane pores) transfection...virus. Virus purification can be a tricky process. You have to centrifuge the culture supernatant in order...The ability to infect non-dividing cells is not restricted to in vitro cell culture as lentivirus-derived...transducing non-adherent cells you might need to centrifuge your cells with the virus in order to aid viral...

...Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations...that sequence modifications can only be made at restriction enzyme cut sites. The lambda red system is an... of DNA within E. coli and is independent of restriction sites. The lambda red system is derived from ...means cloning occurs in vivo, as compared to restriction enzyme cloning where the genetic changes occur...sections, key steps that differ from traditional restriction enzyme cloning will be explained in greater detail...screen for positive clones in most cases, and restriction enzyme digest can be used to screen plasmids ...

...This post was contributed by Kurt Swanson a structural biologist and protein engineer currently working...R206H point mutation on the receptor ACVR1. ACVR1 triggers appropriate bone regeneration in healthy patients...mouse models. As this biologic moves into clinical trials, there is new HOPE for AJ. But, as we in the scientific...fight against FOP in two ways. First, Addgene is distributing ACVR1 Plasmids from a variety of research labs...will be hosting the AJ’s Boston League team (Contributing many new heroes to the team in the process) ...

... a team to get a lot of stuff done.  Factors contributing to job satisfaction Being a good manager is ...mostly happy at work, they will work harder and contribute more. Assuming basic needs are being met (i.e...pay and benefits to live on), the factors that contribute to job satisfaction are a bit surprising. There...overwhelmingly agree that these are the factors that most contribute to job satisfaction: A feeling of being appreciated...organization and feel the importance of their contributions. Flexible work schedule Flexibility to manage...

...shut-down and the subsequent fluctuations in distribution. I don’t have space in this blog to call ...scientific community.  With the dramatic reduction in distribution, we had to figure out a way to maintain our ...operations until our normal work resumed. Addgene distributes and fulfills requests for scientists doing almost...especially proud to support Addgene now, when their distribution of plasmids for COVID-19 research is accelerating... cloning grade DNA service, and viral vector distribution is recovering from the pandemic-related slowdown...

...Human whole genome sgRNA iBAR library Article contributed by Susanna Bachle Figure 1: Initial steps...labeling using recombinase competition Article contributed by Aliyah Weinstein Sparse Predictive Activity...reporter for calcineurin activity in yeast Article contributed by Leah Schwiesow Calcineurin is a protein phosphatase...protein fusions as a DNA staining dye Article contributed by Jennifer Tsang Figure 4: Double stranded...single molecule-fluorescence assay to monitor restriction enzyme digestion of single molecules in real-...

... This post was contributed by guest blogger James D. Fessenden, an Assistant Professor at Brigham and... site when used in conjunction with Cy dye-nitrilotriacetic acid (NTA) conjugates (Kapanidis, Ebright,...50-69. Royal Society of Chemistry. 3. Ermolenko, Dmitri N., et al. "Observation of intersubunit movement...-mediated fluorescent labeling with (Ni2+: nitrilotriacetic acid) n-fluorochrome conjugates." Journal ...

...immune response from the body. Several factors contribute to the immunogenicity of a drug, including the... body recognizes a drug as a foreign antigen, triggering a humoral immune response. ADAs can inactivate...one of the more common open-access tools, while EpiMatrix is a commercially available algorithm to evaluate...in silico assessments, as well as analyze the contributing factors that in silico prediction algorithms...monocytes. PBMCs can be further differentiated into dentritic cells or macrophages through cytokine induction...

...In addition to single plasmids, Addgene also distributes pooled plasmid libraries containing hundreds,...versatile offerings! We recently re-amplified our distribution stock of the Brunello Human gRNA library, and...processes we use to ensure high-quality library distribution. You can also use these tips as a starting point... scaled to 1, to create a “cumulative wealth distribution” curve, or Lorenz curve. By calculating the ...gain a sense of the uniformity of the library distribution, with an AUC of 0.5 representing perfect uniformity...

...presentation, starvation or the use of caloric restriction mimetics trigger autophagy and, in a mouse model led ...deposit and distribution service! References: 1. Pietrocola, Federico, et al. "Caloric restriction mimetics...from a state of chronic hepatitis caused by hypernutrition and alcohol consumption. Chronic inflammation...

...Light-mediated block of synaptic transmission Article contributed by Klaus Wanisch  Listen to the optogenetics ...imaging of microbes in mammalian hosts Article contributed by Lukas Morgan  Listen to the acoustic reporter...and create ultrasound contrast. At a very low volumetric density and with a resolution of less than 100...mechanisms of miRNA-mediated gene regulation Article contributed by Michael Lemieux  Listen to the miRNA reporter...vivo using the CRISPR-Cas9 system CHIME Article contributed by Aliyah Weinstein  Listen to the CRISPR podcast...

...This post was contributed by Mary Tamer from BioBuilder, an innovative nonprofit bringing the study of... new for themselves and when they effectively contribute as part of a team.” As a seasoned educator, Kuldell...almost every state in the U.S. and in more than 30 countries around the world. Snapshots of the BioBuilder ...open-access curriculum begins with students reading comic strips and watching animations that present a specific... lab materials and reagents such as droppers, centrifuge tubes, circuit parts, and lab-strains of bacteria...

...collaborative group, she also joined early on and contributed her expertise in developing reproducibility workshops...Joanne Kamens, Addgene also had the opportunity to contribute to this open science collaboration. The first...workshop at the conference, but because each contributor made the materials accessible, anyone could present...present the material. For example, Addgene’s contributions by Tyler Ford included reagent sharing and were... upcoming conference or you’d like to join or contribute to this collaboration, please check out the official...

...worry about and that is something to recognize! Distributing plasmids, viral service, and more For the ...about succeeding at our core mission. Plasmid distribution is amazingly still growing at >9% every year...since 2005. We’re meeting the challenges of DNA distribution to ensure scientists everywhere can access plasmids... it. Almost 10,000 viral vector samples were distributed in 2018 and we are poised for more offerings....Addgene. There’s so much potential for service and contribution! Leadership On an organizational level, ...

...He has recently developed a third-generation intensiometric glutamate sensing fluorescent reporter (iGluSnFR3...to identify cell populations in the brain that contribute to the development of behavioral signs of opioid...connectivity patterns of these cells and the functional contribution to a variety of behaviors related to addiction...expressed in a mammalian cell line engineered to be electrically excitable. The mutants are co-expressed with...membrane voltage,” says Tian. Figure 5: Citrine-tagged voltage indicator expressed in mouse cortex...

...The post was contributed by guest blogger Londa Schiebinger, PhD, Hinds Professor of History of Science...the US Women’s Health Initiative Hormone Therapy Trial, for example, found that the study saved lives. ...was $35.2 billion (or $140 per $1 invested in the trial). The goal of gendered innovations It is crucially...research is minimal compared to that of human clinical trials. And, as Janine Clayton points out, late-stage ...because sex was not analyzed) may harm humans or contribute to failures in drug development – costs far greater...

... by a variety of methods such as ultracentrifugation, centrifugation on a sucrose gradient, spin columns...producing and using lentivirus and offer some tips and tricks for overcoming these hurdles. Talking 'bout my...relatively inexpensive and does not require an ultracentrifuge, PEG will precipitate all proteins in a solution...high-titer lentivirus preparations without ultracentrifugation. Sci Rep. 2015; 5:13875. PubMed PMID: 26348152...

...they are transferrable across most, if not all, industries and work environments. This is why they are called... bathroom mirror pep talk, forgotten and then retrieved your lucky socks, persevered through a nerve wracking...potential teammates want to be assured that you will contribute openness, clarity, approachability, and professionalism...interview, check out this article from The Muse. Kai Strickland is a former Customer Support and Operations Manager... Kai Strickland...

...This post was contributed by Patrick Miller-Rhodes from the University of Rochester Medical Center.  ...guest blogger Patrick Miller-Rhodes from the University of Rochester Medical Center! Patrick is equal parts...in large enough quantities (and the correct stoichiometric ratios) to generate useable data. What’s more...

...dependencies they identified and their associated ancestries. While there were some exciting relationships...Dependency Map) than lines from European or East Asian ancestries. These SNPs, however, were not accounted for ...racial bias. It is easy, even unintentionally, to attribute to an ancestry group what is actually an artifact...and references References Germline variation contributes to false negatives in CRISPR-based experiments...experiments with varying burden across ancestries. Sean A Misek, Aaron Fultineer, Jeremie Kalfon, Javad Noorbakhsh...

...generally referred to as Antigen Retrieval. I chose not to get into antigen retrieval here because it’s not quite...permeabilization reagents are detergents, such as Triton X-100, Tween-20, or saponin. Each detergent has...membrane-associated proteins in place. In contrast, Triton X-100 and Tween-20 are examples of non-ionic detergents...Permeabilizing with a non-ionic detergent, like Triton X-100, can lose membrane-associated proteins, but...

... This post was contributed by Ryan Watkins, Professor at George Washington University and developer of...may also benefit, such as researchers in other countries or ot­her disciplines, are even less likely to...research from other disciplines, or from other countries, to explore new connections to our research. So...hours of video were watched each month, in 39 countries, and in over 54 languages. While most scientific...citation rates to measure the impact of their contribution. It offers a 5-step process and several examples...

...controlled by collections of neurons that are intricately wired into circuits through synaptic connections...weakly expressed. As these epitopes tags have no intrinsic visual signal, they cannot be detected directly...neurons. As compared to epitopes tags, FPs are intrinsically fluorescent and can therefore be used in live... interactions. Also, overexpression of FPs can trigger protein aggregation which is toxic for the cells...darkened chromophores. These probes are both intrinsically fluorescent and can be detected with highly ...

...This post was contributed by Kusumika (Kushi) Mukherjee, a Postdoc at Massachussetts General Hospital...patients with skin disorders for research and clinical trials, as it allows access to a larger number of willing...phenomenon has been recorded in past gene therapy trials and remains a safety concern in the clinical field...Furthermore, working with Sendai virus requires stringent biosafety measures, thereby increasing the costs...Using Serial Plating of Reprogrammed Cells by Centrifugation. Stem Cells Int, 2016. 2016: p. 1329459. PubMed...

...This post was contributed by Katherine Rogers, a postdoctoral researcher at the Friedrich Miescher Lab...processes—not just development—are available from strain distribution centers in the US, Europe, and China. In addition...pathways—such as those regulated by one-eyed pinhead—contribute to the “sculpting” of tissues and organs during...Katherine W. Rogers is a postdoctoral researcher in Patrick Müller’s group at the FriedrichMiescher Laboratory...El-Brolosy, Mohamed A., et al. "Genetic compensation triggered by mutant mRNA degradation." Nature 568.7751 (...

... Weakly electric fish are excellent systems to study for convergent evolution because electric organs ...leave them to their own devices. Chytrid fungi (Lillian Fritz-Laylin) Chytrid fungi are useful for the study...but there are few genetic tools currently for terrestrial frogs. Cnidarian-Dinoflagellate symbionts (Virginia...

...It is also worth noting that plasmids can be symmetrically incompatible, meaning that both plasmids are... from the cell with the same probability, or asymmetrical, where one plasmid lost from the cell at a higher... higher rate than the coresident plasmid. Asymmetric plasmid loss occurs for several reasons, including...high copy incompatible plasmids that lead to asymmetrical plasmid loss of the usually large, low copy ...: 15916596 Kamruzzaman M, Shoma S, Thomas CM, Partridge SR, Iredell JR (2017) Plasmid interference for...