...of naked DNA. Other investigators who had been working to develop high efficiency gene targeting approaches...the addition of gene-specific 1 kb homology arms flanking a FLOXed neoR gene. pAAV-SEPT-Acceptor, the preferred... CRISPR-based approaches for the generation of knockins via homologous recombination with a homologous...application - the creation of sequence-specific knockins - for which the AAV approach is most well suited...suited, since it routinely generates knockin efficiencies of 1-40% with G418-selected clones. However, there...approach requires G418 selection of infected clones, making it unsuitable for most in vivo applications.  Third... CRISPR-based approaches for the generation of knockins, it is not always ideal. Because of these limitations...

...epigenetic silencer Recombinant antibodies for marking astrocytes MagIC-Cryo-EM for sample enrichment...minimizes sample loss while also supporting imaging, making it easy to identify dozens or hundreds of particles...molar ratio) with 10 minutes of glutaraldehyde crosslinking: representative micrograph (C), particle orientation...could be overcome with methods such as chemical crosslinking or tilted-stage imaging. A truncated form of...compatible with the buffer additive MgCl2 and the crosslinking agent glutaraldehyde. Adding RvLEAMshort to ...j.cell.2021.03.025.    Recombinant antibodies for marking cell types in the nervous system By Ashley Waldron...filament expressed in astrocytes throughout the brain, making it a popular target for labeling these cells. Check...

... pathway. Those can include cytokines and chemokines; enzymes like kinases, phosphatases, and ubiquitin...false positive signal. Dump gating When you are working with heterogeneous cell mixes, like bulk lympho... buffer solutions. Fixing causes protein cross-linking, killing the cell but preserving the cellular state...gating uses the same color for different markers, making it easy to gate on the negative cell population...investigating negative regulators of T cells and cytokine signaling in tumor immunity. When he's not in ...

...in vitro kinase reactions. Often, however, researchers lack knowledge of the regulatory kinase, access ...access to active kinase for the reaction, or have difficulties with kinase specificity in vitro. The establishment...phosphorylation sites. If knowledge of a candidate regulatory kinase is known, it’s possible to generate non-phosphorylated...phosphoserine into recombinant proteins, such as the kinase MEK1 (5). MEK1 is inactive without requisite dual...create a pSer/phosphomimetic dual mutant) elevated kinase activity over 70-fold over dual phosphomimetic ...

...every day, sharing ideas for the company, but also talking about sports, movies, and just about anything. ...cite lunch as one of their favorite parts about working at Addgene. We’re a small non-profit, so we can...Chef-style competitions Every few months, we’ll have a cooking competition featuring a selected ingredient. Anyone...cook-off last week, and the featured ingredient was pumpkin. Everyone gets to vote for their favorites, and...the stiff competition or says something about my cooking.   This is a creative (and delicious) team building...work better and keeps us smiling even when we’re packing the 400th plasmid that day.   As we head into Thanksgiving...

...Does this sound familiar? Well, when it comes to working with Addgene, we certainly hope not! One of our...email. You need help right at the moment you’re looking for it, or you may just prefer to troubleshoot ...icon, search for answers, and choose a result. By clicking on the result links themselves right from within...ll navigate to articles answering your questions making it easier than ever to get the support you need...some reason you can’t find the answer that you’re looking for, or you just have a general question or concern...valuable feedback. This is Addgene after all, and making your research easier always comes first. Resources...

...fused to calmodulin (CaM) and the chicken myosin kinase calmodulin binding region RS20. When calcium is...place for the biosensor to be expressed. Are you looking at an intracellular or extracellular molecule? ...extracellular glutamate sensor pCMV(MinDis).iGluSnFR. Picking your reporter Don’t forget about the other end ...with your lab and experimental setup? Consider what kind of instrumentation you have available, and the conditions...and multi-color imaging experiments. Finally, what kind of system will you use to detect your fluorescent...method depends on what type of information you are looking for. Below we have summarized some of the common...

...its value. My master’s strengthened my critical thinking and problem solving abilities, increased my technical...writer,” among others. Don’t discount job postings looking for PhD applicants since many will also accept ...people describe their actual work. To do this, try looking at LinkedIn profiles of people who have masters...Is their career path one you could see yourself taking? If so, consider setting up an informational interview...degree prepared them for their career.  2. Are you looking for career advancement and growth? A master’s is...project.  3. Do you prefer being a generalist? Working on a variety of projects and in different fields...

...equipment acquisition, public speaking and personnel management?  If you are thinking about a non-academic path...advisors learning how to be a scientist. It is shocking how little pre-work most PhD students and postdocs...learn how to be a scientist. Your PI should be the kind of mentor that will teach you how to: interpret ...with the potential advisor and don’t be shy about asking hard questions. Talk to as many lab members as ... time with everyone. Additionally, if you are looking for more resources to help you choose a good mentor...

...have an opportunity to improve my networking with mentors who are working in USA and Europe, something that...who migrated from their country some years ago looking for their career improvement) and bright students...world in flux. The professional development and networking resources offered by programs like IMP USA-Europe...Institute of Technology, Boston University, Johns Hopkins University, the Salk Institute, and the Karolinska...and contacts with Spanish laboratories to start working on projects together. List of discussion topics...Addgene Read Other Career Blog Posts Read the Networking for Scientists Series Read our Blog Post about...

...Welcome to our Summer SciComm Series, where we’ll be talking about science communication, best practices in ...science communication research.   This summer, we’re making time to talk about my favorite thing - science ...but the foundation of all good communication is making sure everyone’s on the same page. So for our first... endless.    Activity Example audience  Talking about your career as a scientist underrepresented...enjoy listening to podcasts A series of Tweets breaking down a new publication that could advance treatments...Communication - 9 Ways to Get Your Foot in the Door Taking Science to Elementary and Middle Schools   Image...

...cell membrane.  Picking the Right Antibody  It can get even more complex if you’re looking for proteins ...keep epitope availability in mind whenever you're working with antibodies.) Conformation changes usually ...Processes  If you’re feeling relieved that you’re working with native proteins instead of denatured ones ...are interacting with one another, or if you’re looking for a protein of interest while it’s in a transition...validated for ELISAs, it has a good chance of also working for a Western because they both typically use denatured...

... sponsors a global competition for researchers working on rare diseases, offering them the chance to win...Claudio Aguilar (right) at Purdue University and is working to understand Lowe Syndrome, a devastating genetic...great for us,” Wang said. “We were planning on and looking at a bunch of different strategies with CRISPR-...hearing loss and changes in coloring of the hair, skin, and eyes. The researchers found that seven patients... budget is approximately 20,000 USD and we are working on several genetic diseases at the same time. The...studies.” Thank you to all the BeHeard Awardees for speaking with us! Additional Resources Find out more ...

... to email strangers asking to meet. I have cold emailed hundreds of people asking for meetings to discuss... carry a heavy price tag, especially if you're working on something other than software.  If you are a... your entire career in science, you are likely lacking the necessary business background and/or connections... Start with your network You've spent your life making connections and finding mentors -- well, it's time...discuss each project and business decision before making it, to have an internal review system.  On top ...

... and high resolution functional tracking of T lymphocytes, too, making them a versatile tool for application...Max Planck Institute for Neurobiology has been working on genetically encoded indicators of calcium and...has a particular advantage for observing events taking place inside cells that are on the move, such as...element-binding protein (CREB) in living cells. “Now we are making this really good,” Griesbeck said. “We know CREB...genetically encoded magnesium sensor, the first of its kind. “I hope by depositing at Addgene, people will start...

... optogenetic tools in a deep part of the brain, making it sensitive to light. When they illuminated this...patients? Over a quarter of a million people have some kind of neural implant already. If we can use light to...maybe we could treat patients suffering from Parkinson's, chronic pain, deafness, or other conditions.... emotions, to see how a monkey changes decision making, to cure a human patient. We can't do it all by...you have any special tips or tricks for someone looking to try these optogenetic tools out for the first...always tell people, 'Be driven by your science.' Looking for Optogenetics Plasmids? Plasmids for Optogenetics...

...gene edit. I wanted to inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells...Vinculin is a loading control. 3. In terms of making the monoclonal line: I had never done it before...found here. Figure 3: A surprise for the author. Checking Cas9 expression Overview: By Western blotting ...difficult part of the assay was designing primers flanking my edits in genomic DNA and doing PCR on genomic...agarose to get sharper bands. Conclusion Overall, making a human cell line with a gene knockout was straightforward...who you are, you could quickly be on your way to making new scientific observations. References 1. Rago...C, Vogelstein B, Bunz F. Genetic knockouts and knockins in human somatic cells. Nature Protocols 2007;...

...like multi-color imaging (Figure 2). If you’re working with purified proteins in vitro, you can even use...tags makes updating your labels easy! Instead of taking the time to clone a new construct for every new-and-improved...time points.   Figure 3: Single-molecule tracking of actin filament assembly-regulating proteins...developed over the years; improving the labeling kinetics or the fluorophore’s microenvironment to enhance...A., Juillerat, A., Heinis, C., Corrêa, I. R., Kindermann, M., Beaufils, F., & Johnsson, K. (2008). An ...Reinstein, J., Hiblot, J., & Johnsson, K. (2021). Kinetic and Structural Characterization of the Self-Labeling...Selecting Fluorescent Dyes and Ligands GCE4All: Making Genetic Code Expansion Accessible Addgene's Genetic...

...something difficult, be excited about the prospect of working toward a solution. At the end of the meeting, make...person to add a bullet or two about what they are working on. Building this into the agenda ensures that ...items, and Accountability Agendas: Send around talking points for the meeting a day in advance. This gives...Accountability: At your next meeting, start by checking in on the action items from last meeting. Use ...project moving. Use time wisely Meetings are for talking, not reading: Send around relevant documents, like... Executive Director Joanne Kamens FInd Tips on Picking a Research Project Read Other Inside Addgene Posts...

...the day you defend to start thinking about your next step. Start thinking now. Start browsing job postings...in your resume. If the job posting says “French-speaking is an asset,” mention “speaks French.” Employers...groups? Perhaps you had some supervisory experience working with undergraduate students? On top of that, perhaps...universal. Some universities even have officers working specifically to assist graduate students in job...postings and salary expectations public, so start by looking at these university postings to give yourself a...

...you build your lab team and foster an effective working environment? Tom Ellis was offered a start-up package...ideas, and ways to improve the lab. “An effective working environment for research is totally dependent on...communication amongst the team so getting everyone talking to each other cannot be undervalued.” Connie Cepko...of slowly building up your lab and selectively picking the first couple of hires to build a strong, collaborative...resources for Early Career Scientists, including their "Making the Right Moves: A Practical Guide to Scientifıc... Dr. George Church (Harvard Medical School) for taking the time to speak with us about adding new scientists...

... these files to your working directory (the folder that you are currently working in) every time you use...notebooks just won't cut it for bioinformatics. All kinds of biologists are finding themselves using computational... task will keep you from repeating your work or making similar mistakes in the future. Stay organized...comment in C++ and Java Command line history: Tracking command line history by recording all commands...system that does is through trial and error, and by asking for tips from others. Comment below to add your...

...Blog Mentoring for Scientists Series. I have been thinking a lot about Mentoring for over 10 years. Many ...Cepko to learn about her mentoring style. While working on this project, I read the book “Every Other Thursday...Form your own peer mentoring group In my travels speaking to grad students and postdocs I have started to...example, a group of people who are all interested in making a transition. Schedule your first meeting and get...breakfast once a month. All participants must commit to making the meetings a scheduling priority (and last minute...career paths and get their advice. Attend a local networking event together and help each other practice meeting...

..., consider making you own DNA ladders with these plasmids from Penn State Need help picking the right ...cells due to their IgGκ signal peptide sequence, making them easy to harvest from cell culture media. Their...HA staining is weaker than reporter staining. Picking the right reporter for your RANBody While all three...staining GFP, RFP, H2A2B   Are you ready to start making your own RANbodies? Great! All of the RANbody plasmids...learn more about nanobodies Additional Resources Looking for primary monoclonal antibodies to use with the...

... Research. On December 1st 2016, many postdocs working more than 40 hours per week could expect to see... salaried workers receive overtime payment for working more than 40 hours per week increasing from $23,660... postdocs were at institutions focused only on tracking hours, and 22% of postdocs were at institutions...going ahead with their planned raises, some are taking the opportunity of the lapsed mandate (or are being...University of Massachusetts Medical School. We are working with postdocs at other institutions to see if we...that many postdocs already made plans and budgets working around issues such as childcare using money from...

...heavily with other skills such as critical thinking, decision making, creativity, or data analysis. Conveniently...researchers do learn MANY transferable skills while working in the lab and by offering advice on areas where...you spend time in a lab learning techniques and taking on independent projects, you will inevitably develop... skills passively. But I have always found that taking a conscious or active approach can be hugely helpful...are, while also practicing systematic, logical thinking.  If you are already a lab veteran at this point...

... be useful. Mainly because, well, antibodies are kind of hard to see. To solve this issue, researchers...post, where you can find some additional tips for working with these tools.          Enzymes An equally, ...properties allow for efficient signal amplification, making biotin conjugated antibodies good options for detecting...lead perfectly blissful, productive lives without thinking about these chemical reactions. After all, there... are ready to use off the shelf. So why are we talking about this? Even though there are so many commercially...antibodies’ performance after the reaction.  If you are looking to conjugate antibodies yourself, there are kits...

...Novartis Oncology.  Bhang did her PhD work at Johns Hopkins and came to Novartis for her post-doc in January...after treatment with EGFR inhibitor, erlotinib. Looking at barcode abundances among multiple replicate ...Pharmacology group at Novartis Oncology, Bhang has been working on using the ClonTracer library to monitor clonal...their own work. She says, “I spend a lot of time packing, shipping, and sharing protocols one by one,” and...is a “really great way of broadly sharing these kinds of reagents.” Here at Addgene, we’re excited to ... to use! Many thanks to Carrie and Novartis for taking the time to talk with us and for providing this...

...the hopes and concerns of these communities. This kind of engagement and two-way dialogue is crucial if...advising writers and directors in Hollywood; and working directly with community organizations, libraries...leadership and communication experience. These included taking extra coursework and attending seminars in bioethics...writing science articles for a lay readership, and working with middle and high school students. One of these...organizations was pgEd. After obtaining my PhD and working in digital academic publishing for a year, I’ve...and in helping you decide whether you enjoy this kind of work. It is also beneficial to hone your non-...

...deposits per month!), manual transformations were taking a lot of time. Enter the Biomek FXP! We wanted ...improve our transformation protocol and, after looking at a few options, we decided on Beckman Coulter...protocol on the Biomek FxP is pretty simple. By clicking a few buttons you can quickly tell the Biomek ...pick up 8 tips and position the tips indepently making it easy to spread out your samples. This can be...scientists share plasmids across the globe, we’re working on ways to send plasmids as DNA as opposed to in...it suits our plasmid prepping needs perfectly. Working with Qiagen, we developed a protocol to run up ...

...proteins had to be re-engineered for each gene, making wide-scale gene activation seem next to impossible...activation uses dCas9, a CRISPR protein variant lacking its endonuclease ability, to bind to genes without...there are multiple options to choose from when looking to activate genes across diverse cell lines.   ...activation compared to other CRISPR activators, making it a popular method for gene activation experiments...design (SunTag). This streamlines its delivery, making it a common choice for CRISPR activation. Newer... Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-Guided...

...started working in the pharmaceuticals industry after earning my Master’s in 2016 and then working four ...prepare 1-2 unique questions for each interviewer by looking at their LinkedIn profiles. This can help spark...your 1-hour presentation to your audience Start by asking the hiring manager or recruiter what presentation...improving a process or procedure, solving a problem, or making a key decision. Even deciding to stop pursuing ...sharing your screen and won’t be able to see who’s asking a question. Maybe you ask for questions after completing...

... (and Addgene depositor) Craig Mello. Taking his groundbreaking work in RNAi one step further, his lab...the majority of attendees were scientists still working at the bench. It was a fantastic opportunity to...cell membrane. His recent work shows that they are making fairly remarkable progress on establishing a system...the drug resistantance genes in the bacteria thus making the bacteria sensitive to known antibotics. These... CRISPR resources Read Joanne Kamens' Post on Networking at Scientific Conferences  ...

... gRNA using restriction enzyme BsaI. If you’re working in Drosophila, a two-gRNA expressing plasmid is...outside of their recognition sequence, creating flanking overhangs. These overhangs can be customized to...vectors each contain Type IIS restriction sites flanking the promoter-gRNA construct, but with different...with the appropriate Type IIS enzyme, the unique flanking overhang sequences can link together to allow ...constructs with multiple promoter-gRNA cassettes, making them advantageous for small capacity vectors like... mammalian option described below, plasmids for making polycistronic gRNAs are also available from the... tyr gRNA, which causes hypopigmentation, thus marking cells that have undergone genomic modification....

...mentioned above basically involve taking the Myc1-9E10 variable regions and sticking them onto different scaffolds...tagging strategy, another group of researchers was working on isolating monoclonal antibodies to better probe...used in way more applications.   Fig. 1 Making a Protein Tag (Abridged) (1) Locate a promising...same antibody developed almost 40 years ago? Well…kinda. In some cases, antibody providers are distributing.... 1997; Schiweck, et al. 1997) with the goal of making the antibody more amenable to protein engineering...

...between skin and muscle 25–27 < 2–3 mL (~1 mL per site) Beginner Intradermal Anesthesia Between skin layers...Subcutaneous (SQ or SC) injections are given between the skin and the muscles. The most common injection sites... (ID) injections occur between the layers of the skin. While this could theoretically be performed anywhere...shaved first. Restraint: anesthesia Location: between skin layers Gauge: 26 Volume: < 0.05 ml Skill required...cringe or your experiment will allow you to avoid poking the mice, these “no needles” techniques may come...

...in “crispr” and see what comes up. Before even clicking the ‘Search’ button a new content-aware suggestion...jump directly to the type of information you are looking for in our catalog or educational resources: plasmid... your lab. Instead of starting a new search or clicking through to a different part of the website, you...been deposited? In this example we’re going to be looking for recombinant monoclonal antibody plasmids from...finger protein involved in neurodegeneration in Parkinson’s disease. Knowing the primary investigator where...functionally (e.g. Base Editing) and by target organism. Clicking through to RNA Editing brings us to a page with...

...proficiency.  What kind of content are you sharing?  Sometimes your subject matter will drive the kinds of communications...others will avoid it like the plague. Instead of looking for The One Method to Rule Them All, consider the...embark on a project, do your best to understand what kind of resources - including your time - will be required...Podcasts Medium Easy for audience to engage while multitasking. Easy to host guests remotely. Easy access from...things to be physically interacted with. Public speaking is a common fear. Can be discouraging to build...

...large-scale CRISPR screens in cancer cell lines, looking to uncover genetic dependencies of individual tumors...dependencies across ancestry groups (right).    Seeking an explanation  First, they looked for a biological...unexpected discovery, which didn’t make sense in any kind of biological framework, suggested to them that ...information they needed to begin correcting the issue. Working with James McFarland, Jeremie Kalfon, Javad Noorbakhsh...CRISPR guides mapped to a SNP would be ignored. Working with Lia Petronio and Katherine Huang from Broad...process does not stop there. Currently, they are working with Broad's Genetic Perturbation Platform to modify...

...employees spent this remaining time by elaborately pranking each other, dressing in costume, and holding competitions...the question, “What’s your favorite part about working at Addgene?” Most Addgenies reply with an enthusiastic...collaborators have completed their work thoroughly. It’s kind of a chicken and egg situation- do we work so hard...rose-colored-glasses-esque picture of how everyone is kind and collaborative and all we do is have fun all ... To keep the organizational side of things from taking too much of your time, scroll down for some tips...fall into that rut! We’ve done everything from kayaking, to field day, to relay triathlons, to 5Ks. Many...

...and feel the satisfaction of checking off each step as you painstakingly complete your experiment – one...Addgene Website: http://www.addgene.org/ If you’re working with plasmids and you decide to share them using...of useful molecular cloning protocols, including making LB agar plates, restriction digest of plasmid DNA.../ Labguru is a good place to go if your lab is looking to streamline your lab management and you want ...reagent storage and inventory, managing orders, tracking students and research projects, electronic lab...

...modulating the production of the pro-inflammatory cytokine interleukin IL-1 beta in the dorsal raphe nucleus (DRN... circular corridor apparatus using a camera or tracking software. Locomotion is a highly complex process...reflect elements of drug addiction and craving (‘seeking drugs’).  What’s involved in locomotor sensitization...protected) rather than being out in the open (risky). Taking advantage of these characteristic traits, rodents...reduced anxiety levels and a higher tendency for risk taking. These animals also show signs of neuroinflammation...

...chat is a convenient alternative for asking a quick question or making initial contact, but is less suitable...to look forward to in a technical support role Talking to customers everyday, it can be fascinating and...right place to live it out, but ideally without asking too many (annoying) questions. Identifying and ...understanding of when to use these communication skills by working with people from many different backgrounds and... other roles in industry. Many thanks to Eric Perkins and Stephanie Hays for images used in this post...

...consider a dual-sgRNA system. This system uses a “nicking” Cas9, in which one of Cas9’s two critical enzymatic...deleting the catalytic residue of a protein or knocking out a very small peptide. In these cases, you ...xCas9 has a PAM requirement of NG, GAA, or GAT, making it a more flexible cutter with the added bonus ... You can achieve your precise edit without even making a break.  Base editors are dead Cas9 (dCas9) or...sites was sufficient to repress transcription by blocking its initiation. In mammalian cells, robust repression...interest. dCas9-based repressors are useful when knocking out a particular gene isn’t feasible. For example...

..., 2020.   Scientists around the world have been making major improvements to CRISPR technology since its...blue light is readily absorbed and scattered by the skin, so it is not able to reach deeper tissues. Additionally... however, can penetrate 5 mm past the surface of skin and is less phototoxic. Using the FAST system, Ye...By fusing the two pdDronpa domains to the sites flanking the DNA-binding cleft of Cas9, the fluorescent...turn on transcription, label chromatin, and study kinetics. Using chemistry to photocage CRISPRs The aforementioned...spatial control of Cas9 cleavage when using a photomasking technique. Last, Hemphill et al. presented data...techniques (Matony et al., 2020). Whether you are looking to activate, repress, or modify a gene, you now...

...model organism in scientific research. Yes, the same kind we use in breads and other baked goods! Yeast is... the study of neurological diseases, such as Parkinson’s disease (PD). PD is primarily characterized by...C. elegans are also self-fertile hermaphrodites making it easy to conduct large experiments over multiple...60% of human genes have an ortholog in C. elegans making it ideal to study basic molecular biology processes...resources check out Addgene’s Worm Expression page. Looking for more model organisms? Check out Part two in...From the baker to the bedside: yeast models of Parkinson's disease." Microbial cell 2.8 (2015): 262. PubMed...

...politics? Science is an apolitical process for seeking knowledge. The process begins with a testable hypothesis...experiments can help support or reject a hypothesis. Looking at the data, scientists then revise their hypotheses...scientists who hold a doctorate degree, are currently working full-time, and have received a research grant in... of the new administration’s actions, including taking down the climate change page on whitehouse.gov,...depending on the number of people supporting it.  3) Linking scientists to one particular political party while...politicians and citizens to include science in decision making, it will be considered a success. Below are resources...

...without restructuring. Positive Points for Trainees - Taking Steps to Achieve a Successful Career Career Advice...various career opportunities through our career, networking, and management for scientists blog series. We...Session focused on finding this information and making it more easily digestible/available. What I viewed...welfare of the many researchers out there who are making great progress on some of the world’s most profound...At Addgene we hope that all parties involved in making academic research a successful and enjoyable path...Blog Post Read Our Career Blog Posts Read Our Networking Blog Posts ...

...delighted to see enterprising early career scientists taking an active part in creating their own opportunities...opportunities for career development and networking. Aside from the importance of the topics to be discussed, ...building valuable skills and meeting new people by working together on the event planning and execution. I...seeing many similar examples of science trainees taking a proactive role in their non-bench training. Grad...all. For grad students and postdocs interested in taking matters into their own hands, there are some easy...

...honing your science communications skills and networking. And on social media, you could be sharing your...Science Communications and Marketing Coordinator Networking “Many, perhaps most of the most interesting jobs...have to create opportunities. If you find yourself asking, "Why doesn't this product/organization/position...position and my team at Addgene was created.”- Eric Perkins, Director of Science Product Management “I knew.... Usually when I see inspiring artists who are thinking about getting into the arts (or any career to ...