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...stop you. Where do you start? Turns out like all good experiments, making AAV starts with some plasmids. .... You just need three plasmids to start making AAV:  the packaging plasmid which contains the structural...between the ITRs is ~4.4 kb. ITRs are inherently unstable due to their secondary structure, palindromic ...palindromic nature, and high GC content. This instability can cause partial loss of an ITR during routine plasmid... Plasmid video!   Now you’ve got a basic understanding of some common AAV plasmid elements! And while...

...towards a career in science communication, an established field of inquiry with which I was not yet familiar...Herein, a good science communicator—with an understanding of how to appeal to a given audience and how... a scientific background and bring with them established interests and areas of expertise. Perhaps the...individuals respond to issues of unsettled science. Understanding just how the public utilizes insights gleaned... ours reflect a growing effort to address this obstacle in its many forms. A short stroll from my office...by the day, it has never been more crucial to understand just how we receive, process and respond to that...

...while refreshing our image, it was crucial for us to stay recognizable and maintain the trust you have placed... A memorable logo DNA was an obvious place to start brainstorming our new logo. It literally underpins...from our blog to our help center. We hope you’ll start to look forward to seeing it on your packages in...of DNA! Plasmids, and so much more Addgene started out as a plasmid repository. Over the years, we...Science is full of logistical hurdles” is a truer statement of the barriers Addgene tackles, but doesn’t sound...nice. We had a breakthrough when we realized that starting with friction wasn’t true to Addgene. Instead,...

...Tchetchelnitski V, Mehmet H (2012) Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia...culture, only the plasmid with the antibiotic resistance gene and origin will replicate, and the transgene...recombined vector backbone contains the antibiotic resistance gene (AmpR) and origins of replication (ori and...Recombinase-deficient strains like Stbl2, Stbl3, and NEB stable E. coli reduce the rate of recombination in samples...CS, Esposito D (2007) Improved recombinational stability of lentiviral expression vectors using reduced-genomeEscherichia...

...Addgene Blog? Fill out our guest blogger form to get started!   Thanks for all your hard work!   Fluorophores... Nucleic acid based machines Recombinase-based State Machines Enable Order-dependent Logic in vivo Nathaniel...received by a cell. These so-called recombinase-based state machines could potentially be used to report upon...this post to learn more about recombinase-based state machines and how they function. Adapting Toehold...important things to consider if you’re getting started with them. CRISPR Pairing CombiGEM and CRISPR ...efficient genome editing - all influenced by an understanding of Cas9 biophysics! Casilio: An Adaptive, Multitasking...

... use since the 1980s. “Synthetic dyes were the standard in the field, but there is one problem: how to...biochemistry of living neurons in an effort to understand the connection between molecular- and cellular-level...elegans, and zebrafish – essentially all of the standard organisms for which extensive genetic tools are...presence of a ligand (they stick together in one state, but not the other). “It’s quite a robust way to.... “I hope by depositing at Addgene, people will start using them,” Merkx said. “For us, it is one of the...developed something useful [enough] that other people start using it. For us, it’s like a test.” References...

...use grows across the life sciences and to establish standardized validation techniques for academia, industry...both Surveyor™ and Sanger may not be adequate standards for validating edits.  Mismatch cleavage assays...Tsiatis et al. 2010). As the field moves toward standardized thresholds for validating CRISPR experiments...breaks Prediction algorithms: A good place to start for biased validation At the moment, many software...sequences of the genome and sgRNA. This is a good starting point for most researchers as it provides a list...approval. It is key to maintain a consistent set of standards as the field seeks to generate reproducible, quality...

...cells treated with MG-2I dye and exposed to light stained dead within 30 minutes of exposure. 90 seconds ...killing of dL5** cells without death of neighboring bystander wild-type cells.   Zebrafish experiments with ... 2). Induced cell death was confirmed by TUNEL staining.  The video below shows what happens to the zebrafish...as Mg-2I-dL5**. Mg-2I-dL5** also had higher photostability than KillerRed, with KillerRed reaching greater...ablation while having limited collateral damage to bystander cells. Are you ready to use MG-2I-dL5** with your... D. M., Britanova, O. V., Yanushevich, Y. G., Staroverov, D. B., Chepurnykh, T. V., . . . Lukyanov, K....

...suppression (and most everything in between), understanding how, when, and where TFs work is of broad interest... 3,500 human TF isoforms, to help researchers understand this regulatory network. The transcription atlas...fates. The atlas of directed differentiation To understand how each human TF can contribute to differentiation...endeavor, a map of possible TF-induced cellular states was generated (Joung et al., 2023). Fig....screening approach can not only lead to a better understanding of disease onset, but also better simulate a...is a valuable reference if you are looking to understand a pathway, or just looking for the best astrocyte...

...the community to understand and grow the scientific ecosystem, including understanding how shared materials...  An MTA is simply a contract or agreement that states the terms of use for a tangible research material...process, which eventually led to the creation of a standardized agreement on the transfer of biological materials...with many properties, having well-understood standardized terms makes it a lot easier for organizations...One-Time Plasmid Deposit and Distribution Agreement establishes the terms under which your material will be distributed...

...fascinated by the human body and how it works. I used to stay up past my bedtime, poring over my grandparents’...science made me happy, but it wasn’t long before I started becoming disillusioned with the gulf between what... BSc in Anatomy, the University of Otago had established the Centre for Science communication and I was...in neighbouring disciplines might struggle to understand. Why should we trust something that we cannot...know and believe about the world. It is vital to understand what people know – or, at the very least, believe... the National Academy of Sciences of the United States of America, 114(36), 9587-9592. doi:10.1073/pnas...

...You’ve prepped your DNA and you’re ready to get started on the next step of your experiment. But in many...through a liquid to determine the concentration of substances in the liquid. Molecules absorb different wavelengths... methods, fluorescence-based methods require a standard curve, a set of samples with a known DNA quantity... a fluorometer to quantify DNA this way. First, start by pouring your gel containing a DNA intercalating...microarray studies and is not used as much for the standard plasmid prep. Diphenylamine method Another absorbance...made in the visible range and can be read with a standard ELISA reader if no other instruments are available...

...says Bachle. (For presenters, there’s also less stage fright!)And depending on the conference, the audience... questions,” says Zhang. “Usually the moderator starts by asking the speaker the highly upvoted question... the app or virtual platform and making sure you stay connected. If possible, test out the technology ...conference begins.“There were some concerns of having a stable internet connection,” says Abitua about her workshop...chats between sessions or waiting for a session to start. “There was not much time to network,” Zhang says...discussing at tables and the event hasn't even started yet! @neurosymp will be interesting this year. ...

...of this workshop? What do I understand already? What don’t I understand? How does the information I’...prevents it from forming concatemers later. The "FS" stands for Fragmentation System and this is the most important...libraries from small amounts of DNA i.e 100 pg. At the start of the workshop, NEB gave a brief presentation describing...hoped, we found that regardless of how much DNA we started the process with, we got great amplification of...is extremely important for us as a company to constantly update our processes and get new ideas from learning...

...worked with many Addgenies to help improve the understanding, curation, and explanation of our CRISPR resources....” Doench’s graduate work helped expand our understanding of how miRNAs regulate their targets and what...screens to answer their own research questions. Starting at the Broad Institute For his post-doc, Doench... technology development alongside it.” Doench starting working with David Root in the then RNAi Platform...left him a bit skeptical of the technology at the start of the CRISPR craze, but he says, “In the case of...Involvement with Addgene Although Doench’s thorough understanding of CRISPR is what got him involved with Addgene...

... red channel fluorescence images of C2C12 cells stably expressing pTRE-Tight-MitoTimer and rtTA. The cells...shRNA. This system requires less work than using established methods and minimizes some of the technical issues...the CRUSH vector in transgenic mice, the lab established a dual-color RNAi “sensor” mouse strain in which... mapping and electron microscopy A thorough understanding of complex biological systems requires both ... found at Addgene, so what are you waiting for? Start building up your own designer bacteria. St-Pierre...number of libraries containing pre-constructed standardized modules. In the first assembly step, compatible...Nicola Patron have assembled two collections of standardized genetic modules compatible with the MoClo system...

...don’t have a background in statistics and programming, you’d be wise to start working on these skills. ...techniques from across different disciplines to better understand neurobiology and human cognition. Developing ...interaction between neuroscience and other fields. For instance I met one researcher studying how the microbiome... apply your knowledge of metabolism to better understand how aberrations in brain biology lead to altered...always suggest a plasmid to us, request that we start making the AAV, or help us produce new educational...

...practices (QRPs) can quickly lead us to find statistically significant results that are not robust. Sampling...Preregistration is the process of creating a time-stamped research plan in a public repository before conducting... is typically done right before data collection starts, and should include a specific plan for how data...each hypothesis will be tested with a specific statistical test.  Preregistration makes clear the distinction...to present that work using hypothesis testing statistical tools that generate a p-value.  As an example...sampling noise with registered reports, the gold standard for testing hypotheses Finally, Registered Reports...

...mechanism by which bacteria have gained antibiotic resistance and virulence. Scientists have studied how HGT... Guillemin, 2006) or the presence of specific substances (Meibom et al., 2005). Though not all bacteria... replication-incompetent phage particles with assistance from a ‘helper’ phage prior to transduction. ...expressed but not integrated into the genome and stable cell lines, where foreign DNA is incorporated into...can infer genetic advantages such as antibiotic resistance. Bacterial conjugation as explained by... genetic material can pass through. If you are starting any molecular biology experiment check out Addgene...

.... To get started, you will first need a primer that complements your plasmid sequence. Start with a backbone-specific...plasmid, you’ll need a way to find the needle in the haystack: the one perfect clone that contains the plasmid... lost a specific gene survive (ex: antibiotic resistance marker). In a previous blog post, we covered ...plasmid backbone, as it often relies on antibiotic resistance. But what about identifying clones that contain...Lynne Postovit’s lab. It contains an ampicillin resistance marker in the backbone. A selection on agar containing...