... Listen to Our Segment on Magneto 2.0 Hot Plasmid 3: The Human Kinase Domain Constructs Kit from John ...this new way to learn about plasmid technologies. If you have any comments about the Hot Plasmids Podcast...with these efforts, you’ll find new ways to learn about science careers, lab protocols, and, of course, ...each one, plug in some headphones to learn more about the latest and greatest plasmids through this podcast...

...Featured Topic Page Learn about Non-Homologous End Joining Learn about Base Editing Read 3 Tips to Improve HDR...pathway (Charpentier et al., Nat Com).     Fig. 3: HDR events compete with NHEJ-mediated edits of small... post on increasing HDR efficiency to read more about optimizing this distance. Homology arms and types...screening strategies for your experiment. Also learn about Addgene’s knock-in plasmids for reagents to help...

...genes which help mediate AAV viral replication; and 3) the transfer plasmid which encodes a transgene of...ITR-containing plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours...a workspace with plasmid, not that I would know about this… A no template control (NTC) should always ... variability. Check out this post to learn more about different ways to titer AAV. Do you have any AAV...at Fred Hutchinson Cancer Center for teaching me about AAV production and purification! References 1. Aurnhammer...

...expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more gRNAs Two separate lentiviruses delivering...characterize the ‘social networks’ of 157 super enhancers.  3. Measuring temporal changes in chromatin interactions...with current 3C methods provide useful information about chromatin interactions, they are grainy so it’s ...interactions opens the door for answering more questions about chromatin conformations. The Xu lab presented three...Cas9 Variant for Your Next Experiment Learn More About Designing gRNAs Against Your Favorite CREs Here ...

...with a single insertion because it will segregate 3:1 in the T2 generation. You also have no control over...PMID: 17087829. PubMed Central PMCID: PMC1636043. 3. Kyndt, T. et al. (2015) ‘The genome of cultivated... J. (2002) Arabidopsis : a laboratory manual. Cold Spring Harbor Laboratory Press. Available at: https...https://books.google.at/books/about/Arabidopsis.html?id=IfZAMNPWVk4C&redir_esc=y (Accessed: 21 September ...

...PMID 25282355. PubMed Central: PMCID: PMC4317352. 3. Beisel, Chase L., Ahmed A. Gomaa, and Rodolphe Barrangou...Targeted Bacterial Population Editing.” Cell Syst. 1(3):187-196. (2015). PubMed: PMID 26973885. Additional...organizations are working to help educate the public about proper antibiotic usage. Although these steps are...laudable, they likely won’t solve the growing problem. Above all, we need new antimicrobials, ideally ones for...used to deliver any of the three systems discussed above, with the caveat that phages are very diverse, and...

...assessment of many protein targets in a single sample; 3) producing recombinant, partially humanized rabbit...antibodies to be used as a powerful reagent in the laboratory.   The DSHB focuses on monoclonal antibodies,...Hybridoma Bank (DSHB) since 1996.       More Blog Posts about Scientific Sharing: The 10 Most Distributed Plasmid...

... kb at most - which means that a gene larger than 3 kb cannot be delivered using this vector. For example...emerges from disgrace to be the next big thing, again 3. Acland G. et al., 2001. Gene therapy restores vision...famously visited Congress to help increase awareness about the potential of gene therapy!). It would take another...muscular atrophy, neurodegenerative diseases and metabolic diseases are also underway and reported results...

...31668202421', style='text-align: right;' %} Figure 3. An animated map showing the plasmid orders and new...were zero plasmid requests. Afterwards, from 2/24/20–3/15/20 (the supposed end of lockdown), request activity...in his laboratory. Learn more here. Additional resources on the Addgene blog Learn more about plasmid...

...sequence only functions at the extreme C-terminus [3]. You must therefore determine your cloning strategy...PMID: 19819147. Pubmed Central PMCID: PMC2784028. 3. Munro, Sean, and H. R. Pelham. "Use of peptide tagging...moxFPs over standard FPs for the reasons listed above. In addition, because the moxFPs are highly monomeric...Medicine. Her doctoral research was conducted in the laboratory of Erik Lee Snapp and focused on optimizing fluorescent...

...below: 1 Transfer Plasmid : 1 pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper: 2 RepCap Select what...packaging experiments. Let us know what you think about the calculator in the comments section below. References...

... As a small, tight-knit organization we thrive on 3 things: fun, teamwork, and competition. And our Halloween...need more convincing, I’ve got just the proof: Above our office team shows their love of plasmids! Each...

...propagation along dendrites for the first time (Figure 3). The team also used AAV9 to deliver ASAP5 to the ...microscopy at depths up to 600 µm. Figure 3: ASAP5 for voltage imaging. A) ASAP5 selected for ...synaptic events By Brian O’Neill The Lin Lab and collaborators recently created ASAP5, a faster and more responsive...

...and makes your experimental setup less cumbersome. 3. Portability across multiple systems Terskikh et al...PMID: 19889833. PubMed Central PMCID: PMC2801723. 3. Verkhusha, Vladislav V., et al. "An enhanced mutant...Additional Resources on the Addgene Blog Learn More About Fluorescent Protein Aggregation Catch Up On Your...

...the gel and add gel components to polymerize. Step 3: digest the sample to allow for even expansion.  ...:832–863. https://doi.org/10.1038/s41596-018-0117-3 Wassie AT, Zhao Y, Boyden ES (2019) Expansion microscopy...applications and there’s just not much to be done about that.  Second, expansion introduces distortions....

...been trafficked between locations). Figure 3: A) LOV-Turbo becomes active when illuminated with... h, and PMA activation induces cFos::GFP-iPAK4 at 3 or 6 h. Scale bar 5 µm. Images adapted from Lin et...section of the Addgene antibody page, and learn more about the whole-brain reference atlas efforts, including...

... from and how the idea got off the ground Chapter 3 - Addgene as a leader in standardized tech transfer...repository’s future. If you’ve ever been curious about what it takes to start your own nonprofit, this ...Harvard University, where she studied diabetes and metabolism. Melina has worked at Genetics Institute in Cambridge...

...not immunogenic, a necessity for in vivo studies; (3) is compatible with single gene KO and pooled screening...faithfully model the complex spatial, cellular, metabolic, and cytokine microenvironment that exists in ...LaFleur is a Postdoctoral Fellow in Arlene Sharpe’s laboratory at Harvard Medical School and is interested in...

...the impact of research" (2013) Scientific Reports 3, Article number: 1649 doi:10.1038/srep01649     ...Of course, all of you toiling away in laboratories this holiday season want the work you are doing to... has to do with online accessibility. It is now about as easy to find a paper in a lesser-known journal...

...Improved Vectors for Gene Delivery. Curr Gene Ther 5(3), 299–310. PubMed PMID: 15975007. PubMed Central PMCID...Concepts in the pathogenesis of rabies. Future Virol 3(5), 481–490. PubMed PMID: 19578477. PubMed Central...viral vectors is serotype-dependent. Gene Ther 20(3), 348–352. PubMed PMID: 22418061. PubMed Central PMCID...to its retrograde axonal transport. J Cell Biol 82(3), 798–810. PubMed PMID: 92475. Taymans, J. M., Vandenberghe...usually severely impaired, and only few reports exist about dye molecules that are transported from one neuron...

...up the experiment. The first way, it will take you 3 days to complete the entire experiment, but you’ll...be quite difficult. In my experience, planning has 3 components: A mechanism for blocking off time for...post useful. After a bit of research and thinking about my own processes, I’ve broken down time management...task, you’re actively prioritizing. In my anecdote above, I prioritized splitting cells over meeting up with...was too late. I didn’t spend enough time thinking about the possibilities and how to prioritize them. When...month, or even the year, you should be thinking about two main things: How useful the task is (its utility...plasmids to send to Addgene). Following my advice from above, you should probably take the second option. Even...

...understand your proposal to give you proper consideration. 3. Present an easy to follow seminar See item 2. Attendees...? How will your approach resolve this problem? (2-3 sentences) What will be the focus of your first... potential colleagues study. Think about how you might collaborate or, at the absolute minimum, can appreciate...the first sentence or two what your research is about and what you think you can accomplish. No matter...your seminar accessible. Let the experts ask you about the details and then you can display your deep knowledge...expert's question than to leave everyone confused about your research because you waded too deep into the...simple in principle The first question I usually get about chalk talks is, what is a Chalk Talk? It is a 60...

...limitations. 3. Always provide value to others This is a non-profit organization. Therefore, collaboration and ... of how much you can expect to use in the future. 3. Identify the skills needed to use these tools Outline...a successful negotiation and collaboration. We’d love to hear more about your experiences and opinions...can get away with using computer modeling and collaborations rather than using animals, then that’s a cost...associated with old equipment, like service repairs, labour costs, and loss of time, can exceed the cost of...Everything is negotiable. Try asking for discounts, collaborations, access to internal award applications, etc...Happy Sciencing, Damien   Damien Wilpitz is a laboratory research management consultant and coach in Boston...

...primarily show a protein’s presence or absence. Figure 3: A western blot detecting proteins using the indirect...are often used in clinical applications. Diabodies - Diabodies contain two Fab fragments, each recognizing...cells isolated from animals immunized as described above are used to create hybridomas, which produce large...different epitope, bound by short peptide linkers. Diabodies can be used for assembling protein nanostructures... Includes: Flow cytometry Read on to learn more about the applications common to each category, including...controls, and any special considerations to think about. For step-by-step instructions of many of these ...protein of interest, as described in the IP section above. Reverse the crosslink, isolate DNA, and perform...

...back to the drawing board.  Cycle 3: A Return to HQ Figure 3: Even Blugene is practicing social... Production Specialist, I’m constantly thinking about ways to improve our productions to deliver the best...four months after I started with Addgene, I was about to have a check-in with my boss Jennifer. I had ... would be a couple months. Jennifer and I spent about an hour that morning scrambling. Everything we had...someone to be comfortable on camera while thinking about things like lighting, camera angle, and shot composition... allowing people to sit without having to worry about getting in close to mic them up. Monitor: I then...first video in our Intro to the Lab Bench series about PPE! We made this with a combination of in-lab filming...

...the world — and we really do mean all over (Figure 3). We continue to add new posts every week, and we’...beautifully designed and easy-to-reference eBooks. Figure 3: Global readership of the Addgene blog in 2023. Views...a less tangible form: knowledge and information about the materials in our repository. We started to increase...by making it easier to find materials and learn about them. As early as 2007, we began grouping similar...professors, stopping by to say, “Everything I know about virus, I learned from Addgene,” or “I learned to...seeing our resources shared via social media, on collaborators’ websites, or as a recommendation in a Reddit...updating and expanding our website, we’ve learned about the importance of smaller, systemic changes, like...

...experimental design and gel quantification. Figure 3: In a perfect world, protein concentration in your...versus the amount of protein loaded on the gel (n=3). Data is presented as mean ± SEM. Adapted from Analytical... this chain (blue) to infer or calculate things about earlier ones. In practice, multiple sources of error...condition 2.” By eye, the loading control appears about the same between the two conditions. But it isn’.... However, it throws both assumptions discussed above into question. Assumption #1: The loading control...searching the literature to see what is already known about your housekeeping protein in your context. If you...step from the POI blot, you don’t need to worry about the linear detection ranges of multiple targets....

... originally written by Melina Fan and updated Nov 3, 2022 by Susanna Stroik. You’ve designed your gRNA...PCR and a restriction enzyme digest.   Fig. 3: Restriction enzyme screening for resistivity to digestion... your edit, it’s important to know a few things about your target cells and the desired edits. First, ...et al., NAR). It works just like TIDE (outlined above), except that it uses a 3rd sequencing trace file...great option, especially if the options outlined above aren’t realistic. Check Out Our Post on Sequencing...higher probability locations. If you’re worried about off-target effects, you can consider using a version...SpCas9-HF1, eSpCas9(1.1), or HypaCas9. Read more about Cas9 options in our blog post here. Dealing with...

...conservative estimate. As a graduate student, I drank 3-4 cups of coffee a day, and I had colleagues who were...learn about all the amazing research being done with plasmids. I heard an impressive talk about the use...pulls off such an inspiring, thought-provoking, collaborative event – all while keeping the party going! At...scientists we've spoken with are excited and passionate about making such tools open-access. Though it is time...

... why they are the most commonly requested (Figure 3). Over 400 biosensors were distributed in 2023 alone... viral vectors that deliver GFP.   Figure 3: A) Cumulative AAV preps available at Addgene from...all, a delivery method. I would be remiss to talk about them without looking at what they’re delivering .... But in the years since, we’ve learned so much about what it takes to make these viral vectors not only...., 2016), video, educational resources, and collaborations, and will continue to do so, in the hopes that...leave a comment; or take our AAV survey. Learn more about GCaMPs and other tools developed through the GENIE...

... them. For intracellular targets, we usually test 3 different fix/perms (methanol, PFA-saponin, PFA-triton.... The approach we suggest is summarized in Figure 3. The principle is to confirm the antibody can detect...-use of an individual experiment.   Figure 3: Our recommendations for an approach that may be used...leukocytes. The file shared also contains information about the labeling pattern of several cell lines using...Laflamme, C., Virk, H., NeuroSGC/YCharOS/EDDU Collaborative Group, & ABIF Consortium. (2024). A guide to...

...startups, and real applications of SynBio. Chapter 3: Synthetic biology as the engineering of biology at...synthetic yeast chromosome. Listen to learn more about Dr. Ellis, synthetic biology, synthetic chromosomes...Additional Resources from the Addgene Blog Learn about the Synthetic Biology Education Nonprofit BioBuilder...BioBuilder Plasmids 101: Golden Gate Cloning Learn About Working at a SynBio Startup in Our Interview with...

...negative controls for genome-wide CRISPR screens; and 3) safe-targeting guides, which target genomic sites...PMID: 25513782. Pubmed Central PMCID: PMC4320685. 3. Fu, Yanfang, et al. "Improving CRISPR-Cas nuclease...following unanswered questions about CRISPR screen design. Unanswered questions about genome-wide CRISPR/Cas9...Morgens et al drew some generalizable conclusions about off-target activity, demonstrate safe-targeting ...

... without scrolling up and down the page.  Figure 3: The Education & Tools menu Further down What happens...straight from the new search bar, but the menu just above it can help you quickly and easily navigate to educational...down? You’ll find Blugene, as well as information about Addgene, our mission, links to our deposit, request...

...:02 : Niroshi and Ben introduce themselves 2:03 - 3:22 : How did you end up becoming a graduate student...graduate school community to help keep you excited about coming into lab even when things get tough in the...

...transfer plasmid with the E1 and E3 (Early 1 and Early 3) genes deleted. These genes’ functions are rescued...Degeneration in Tissue Culture. Proc Soc Exp Biol Med. 84(3), 570–573. https://doi.org/10.3181/00379727-84-20714...this introduction gives a sense of what AdVs are about – and we hope that it inspires further use of existing...

...Originally published Mar 3, 2016 and last updated Apr 13, 2021 by Will Arnold. Although CRISPR systems...a subsequent curing protocol that requires only 2-3 hours incubation. E. coli and T. citrea scarless editing...the QCascade DNA-targeting module with the crRNA, (3) three transposase proteins, and (4) the donor DNA...CRISPR Featured Topic Page Learn more about Cpf1 Find out about CRISPR Multiplexing Resources on Addgene.org...coli and other bacterial species. Read on to learn about the tools available for bacteria and some of the...that can be used in this way. As described in the above CRISPRi section, the Marraffini Lab published a ...as a collection of organisms, their genes, and metabolic processes, occupying the gut. Many human diseases...

...228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon, Chun-Hyung Kim, Jung-Il Moon, Young-...PubMed PMID: 19337237. PubMed Central PMCID: 2677165. 3. Jia, Fangjun, Kitchener D. Wilson, Ning Sun, Deepak... deriving human iPS cells." Nature Methods 7, no. 3 (2010): 197-99. PubMed PMID: 20139967. PubMed Central...methods do you have experience using? Let us know about your iPSCs experiences in the comments below! References...Additional Resources on the Addgene Blog Learn about Methods to Reprogram Peripheral Blood Cells Check...

...on the future of the scientific endeavor October 2-3, 2014 Boston University Jacob Sleeper Auditorium, ...doing science in some form? There is a lot of talk about the state of scientist training in the US and around...

...individual, sharp and evenly spaced peaks …like these…. 3. Expect to get 500-700 bases of clean reliable DNA...and troubleshooting your DNA sequencing results Above (top right corner of this post) is an example of...

...are 24 variants of the cas14 gene that cluster into 3 subgroups (cas14a-c). All these variants share the...Additional resources on the Addgene Blog Learn more about DETECTR Browse all of the CRISPR blog posts Download...

...Method with High Throughput Capability." PLoS ONE. 3(11): e3647 (5 November 2008).  ...protein,” he says. “There is nothing you can do about it but be gentle. If it no longer works, just trash...

...between two AAV transfer plasmids, the first with a 3’ splice donor and the second with a 5’ splice acceptor...transfer. Kwon I, Schaffer DV. Pharm Res. 2008 Mar;25(3):489-99. PubMed . Adeno-associated virus serotypes... Z, Asokan A, Samulski RJ. Mol. Ther. 2006 Sep;14(3):316-27. Epub 2006 Jul 7. PubMed . Recombinant AAV...therapy. Grimm D, Kay MA. Curr Gene Ther. 2003 Aug;3(4):281-304. PubMed . Vector genome integration: Adeno-associated...Learn about the types of plasmids used to produce AAV in the lab. Addgene’s AAV Guide will teach you ...you about AAV components, serotypes, pseudotyping, and more. Science...non-homologous sites in the genome, and do so at about a 0.1% frequency. Nonetheless, the majority of rAAV...

... MD but later got obsessed with research. Chapter 3: How all basic research ends up being translational...promote scientific sharing. Listen to learn more about Darrell, his research, and his focus on open science...Medicine and in the Department of Pathology and Laboratory Medicine. He attends in the Medical Intensive...NIH-funded Principle Investigator in the Kotton Laboratory and is the founding Director of the Center for...Podcast.     Additional Resources from Addgene Learn about Retinal Gene Therapy from Connie Cepko Find Plasmids...Plasmids from the Kotton Lab Learn more about Stem Cells ...

...with an isotype-matched control.      Figure 3: A preimmune serum control addresses situations where....” British Journal of Pharmacology, vol. 175, no. 3, Wiley, 19 Jan. 2018, pp. 407–411. Crossref, doi:10.1111...subjected to the experimental condition. In the example above, a negative control could be a third group of mice...in the experimental protocol. In the experiment above, you could repeat the tau western blot several times...Baseline secondary signaling levels In addition to the above-mentioned safeguards, you’ll need to test the secondary...Flow cytometry In addition to the controls listed above, viability and compensation controls are recommended...technique before starting. For more information about antibodies and antibody-based experiments, check...

...working with RNA and extracting RNA without a kit. 3. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR... highlighted reagent repositories and other collaborative efforts in the scientific community to speed...store my plasmid in TE, Tris, or water? And what about temperature? Find the details in this blog post....dominates DNA quantification discussions, but what about the other methods to quantify DNA? Here we cover...Incompatibility by Leah Schiesow If you're thinking about hosting multiple plasmids in one cell, it might ...

...available as viral vectors (here and here).  Figure 3: JEDI-2P was identified from a high-throughput screen...microscopy. Cell. 2022 Aug 16. pii: S0092-8674(22)00916-3. doi: 10.1016/j.cell.2022.07.013 pYtags illuminate...screen (Koveal et al. 2022). Lactate is a key metabolite in glycolysis, so this sensor will be a valuable...valuable tool to study cellular physiology and metabolism under various conditions. LiLac has improved response...going viral! In addition to the viral preps noted above for iGluSnFR3 and JEDI-2P, several biosensors already...innovation next year! Did we miss any? Let us know about your favorite new biosensor plasmids in the comments...

...perform downstream characterization of, isolating 2 or 3 distinct clones to perform key experiments on is a...CRISPR-Cas System in Gene Editing. Mol Ther. 2021 Mar 3;29(3):937-948. doi: 10.1016/j.ymthe.2020.11.009  Guo C,...is an off-target effect. Should I be concerned about off-target effects? Depending on your experimental...this could be a huge concern, or nothing to worry about all. Let’s consider several scenarios below for ...Predicting off-target events As mentioned in the above section, there are gRNA softwares available which...diligence to ensure they are indeed a non-issue. Above we mentioned how to predict off-target frequency...

...Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at...Blog Learn about photoswitchable kinases Learn more about fluorescent biosensors Learn about using phosphoserine...

... guest Drew Endy, in celebration of open science. 3)      Also in June, the G8 Science Ministers issued...study probably doesn’t say as much about open access as it does about the state of peer review. 8)     ...University Open Data Access (YODA) Project, “a collaborative endeavor guided by aspirations to promote open...