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...for students, postdocs, and young Principal Investigators (PIs). We've interviewed Addgene depositors ...Medical School) who took the time to answer our questions.   Advice for success in academia How can we...passion? Even if no one else is excited about it, distinguish yourself with this passion. “Love what you are...laissez-faire; theory or practice; business-like or artistic; tech, anti-tech or pragmatic.” Advice for pursuing..., and George Church for answering our burning questions about how they manage and motivate their labs!...Posts in this Blog Series   Our 3 PIs answer more questions, including: Advice for Choosing a Research Project...

...Massachusetts Institute of Technology, Boston University, Johns Hopkins University, the Salk Institute, and the...USA-Europe not only provide the specific technical and logistic support to increase the chances of success, but...evidence of this, you needn’t look any further than testimonials from the students who participated in the program...common problems that many others have faced yet, still, this challenge did not hinder them from advancing... the Karolinska Institutet, among others). Mentors and mentees held scheduled monthly progress video conferences...universities and high level public and private institutions also showed their support and hope for the IMP...

... write test question banks, case studies, and experiments for labs, in addition to testing those experiments...Labs as part of their curriculum by either taking existing curriculum and customizing it for schools or developing...curriculum development team at eScience Labs. She still gets to write curriculum, but has taken on other...responsibilities.“I’d say maybe 25% of my day is still what I did as an SME, and then 75% of the time I... proposals, a few PhD theses, and occasionally posting something science-related on Facebook. “I think...

...of similar interests and goals. The following questions can serve as a guide when selecting a research...impact on your field? You want to choose a research question that people (especially you!) care about. ...project along the way. Advice from principal investigators Tom Ellis highlights the importance of keeping... apart a piece that can address a topic in a mechanistic way using the technology available.” Choosing... your project will be fresh as well.” Don't underestimate the importance of choosing a solid research ...Posts in this Blog Series Our 3 PIs answer more questions, including: Advice for Choosing a Research Project...

...sharing on the site. The entry form has 5 general question types and it takes approximately 15 minutes or... have all of your data prepared. All of these questions relate to factors that you should have already...re running your next AAV experiment, use these questions as a guide for designing your future injection...information so we can follow up with you if we have any questions and so that we can give you recognition for sharing... strain. Experimental design & methods: These questions are focused on injection and detection methods...data. Please note that the data hub is currently still in beta version, so we’re always looking for ways...

...Leiwe and Takeshi Imai from Kyushu University. Stochastic multicolor labeling is a powerful solution for...microscopy-based neuronal reconstruction. To achieve stochastic multicolor labeling, Brainbow used the Cre-loxP...copies of the transgene cassette are introduced, stochasticity will result in a combinatorial expression of...Brainbow was inherently low. This is because the stochastic and combinatorial expression of fluorescent proteins... such vector-based gene delivery systems, the stochastic expression of XFPs is possible by simply introducing...introducing a mixture of XFP genes. Stochastic multicolor labeling is possible when each of the XFP genes...

...came as an undergrad at Worcester Polytechnic Institute. Though some WPI students chose to go the solo...goal, or at the very least, a clear reason for existing. Here are a few types of teams that exist at Addgene... team A Process Team is a group of people with distinct skills who fulfill a long-term function. When ...biochemist, a geneticist, a microscopist, a biostatistician, and that one golden-handed technician who ...responsibilities are. That’s a subtle but important distinction. If I invite someone to be on a project team,...out the discussion themselves, then one has to question whether that person should be on the team. As ...self-interest ahead of the team’s goals, then that team is destined to do poorly. To some extent, it doesn’t even ...

...don’t get the same result. So, then they end up investing time that they thought would be moving forward... build on it going forward. This is not just a question of being honest and straightforward, it’s an important...do we get where we want to be? The part that is still missing is a clear alignment of incentives. Right... in science needs both a job and funding, so institutions and funders, to a large extent, hold the keys...is clearly room to do more. If we could really instill a culture of rewarding good scientific citizenship...UK and then a postdoc at The Scripps Research Institute in La Jolla, California. She joined Elsevier after...

...study 1: Cell lines Many of the reagents used to investigate SARS-CoV-2 were developed for broad use and are.... Seventeen of these cell lines were used to investigate the susceptibility of cells from different organisms... Bioresources Cell Bank, part of the National Institutes of Biomedical Innovation, Health, and Nutrition...choose the best experimental model for further investigation of this pathogen. The sharing and distribution...The Jackson Laboratory, a nonprofit research institution, has been able to rapidly propagate this strain... rapid pace of SARS-CoV-2 research by removing logistic hurdles to continue making materials available...

...upon who your challenger was, you might have to drastically alter your strategy. I realized that similar ...As your students play, you can observe, answer questions, and guide them to productive strategies while...they had 10 minutes to expose their strategy, justifying each single element used to build the construct...concepts required by the course and some were so enthusiastic that they took the cards and the board with ...modifying BUILD YOUR PLASMID You can find rules, suggestions, and more details on how to play the game on ...biomedical education for future generations. All suggestions, tips and critiques to improve the educational...

...students are confronted with the question ‘what now?’ This is often a hard question to answer if you plan on ...These skills show employers that you can fully investigate and solve a problem or complete a project, overcoming...Manager Patent Lawyer Science Market Research Statistician   Attention to detail Patience, accuracy ...including: Science Reporter Technical Writer Statistician Bioinformatician Science Writer Grant Writer...like for someone who currently in the position. Questions you’ll want to find answers to include: Will ...

...Daley, Christine Mummery, Lorenz Studer, and Deepak Srivastava--addressed a battery of questions regarding...particularly in fields like stem cell biology where questions of ethics can have profound impacts on the development...banked iPSCs across international borders. While questions of ethics can be daunting because they have no...cell community for addressing these important questions that the scientific method cannot address. Listen...already mentioned, Addgene depositors Marius Wernig, Kristin Baldwin, Kathrin Plath, Hans Schöler, and closing...

...repair bias is a fascinating (and yet unanswered) question. Our recent paper (Richardson et al 2016) revealed...that process. We began our research with a simple question: how do Cas9 and sgRNA interact with and dissociate...from a DNA target? The surprising answer to this question led us to develop a new, rational approach to ...followed these experiments with more detailed investigations into the release of DNA post-cut. Using substrate...repair that normally accompanies Cas9 cutting. We still don’t know the mechanism underlying dCas9 editing...Postdoctoral Researcher in Jacob Corn’s lab investigating DNA repair pathways.     Additonal Corn Lab ...

... the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher and requires...requires more optimization. Enzymatic digestion, while gentler on the sample, introduces bias due to the...sonication is usually the preferred method, enzymatic digestion can be appropriate when your protein of interest...cofactor. In such cases, the gentler enzymatic digestion can preserve more of the protein:DNA complexes...After crosslinking and sonication or enzymatic digestion, you’re ready to incubate the sample with your...

...we can’t forget that those other components are still there in real life.  Certainly referring to any ...Chromosome Organization. These aren’t the only investigators in the chromatin field who have deposited with...associated PI pages on the Addgene website. In-exhaustive List of Addgene Depositors Who Study Chromatin...Chromatin Cheryl Arrowsmith James Bradner Kerstin Bystricky Courtney Hodges Sharon Dent Archa Fox Elaine...consider searching directly on our website. In-exhaustive Chromatin-related genes with plasmids on Addgene.org...@theReddyLab), Thoru Pederson, Joanna Wysocka, Kerstin Bystricky, Ben Martin, and Berkley Gryder. Addgene...

...life of its own. Nevertheless, there are still many open questions to be clarified, especially with regard... field, often referred to as 'frugal science', existing components are adapted and combined with creativity...raw data (CARE), segmentation of imaging data (ILASTIK), or the popular plugin-based image process framework... put their work on the shoulders of giants. The still relatively young movement of sharing open hardware...References Joel T. Collins, Joe Knapper, Julian Stirling, et. al, "Robotic microscopy for everyone: the...

...Have you ever tried digesting with XbaI or ClaI restriction enzymes and gotten unusual or unexpected ...site-directed mutagenesis experiment? The answer to both questions is the same--methylation! Read on to learn about...from the restriction modification systems, yet can still affect whether certain restriction enzymes can effectively...potential for methylation should be considered when digesting DNA. Why? Well, even though Dam methylation sites...performing a digest for cloning purposes or for diagnostics, we suggest double checking to make sure your...affected by methylation. This table is by no means exhaustive, so you may want to consult the REBASE database...

...toxicity, it would be advantageous to instead use a preexisting, conserved cellular machinery to process gRNAs...any 5’ leading nucleotide could be processed, a distinct advantage over other methods that favor certain...indicating that Cas9 can be guided to at least 8 distinct loci. They also examined editing efficiency in...multiplexing in systems with limited packaging capacity. Still confused about the various tRNA expression strategies...systems available from Addgene. If you have any questions or thoughts about this technology, leave them ...them in the comments below.  References Boosting CRISPR/Cas9 multiplex editing capability with the endogenous...

...synthetic biology is still in its infancy. A long timeline from transformation to testing and a lack of well-characterized... of plants are being investigated on limited funding and time. While institutions that focus on creative...problem solving like the Howard Hughes Medical Institute have begun to pivot towards plant biotechnology...Biophysics and Biochemistry from the Illinois Institute of Technology in 2004, she accepted a commission...

...varying degrees of β-arrestin-2 recruitment to receptors, indicating their agonistic nature. The potency...microscopy techniques, such as Stimulated Emission Depletion (STED), and Stochastic Optical Reconstruction Microscopy...help. Here, we will outline some fundamental characteristics to consider when selecting a dye or ligand ...Fluorescence microscopy takes advantage of the characteristics of these fluorophores to enable highly specific...non-fluorescent states in response to a specific stimuli, which leaves only a small volume of fluorophores...

...appropriate containers. The machine is useful for distinguishing cell populations that have been tagged with...visualize how cells interact after a particular stimulus. Using an epifluorescence microscope and GFP fusion...easily track GFP expressing cells. For example, by harvesting lymph nodes at different times after antigen ...immunologists the ability to count cells and statistically analyze cell movements. By using cells labelled...immune response, but the study of these dynamics was still laborious as epifluorescence microscopy wasn’t able... from Kamenyeva et al. 2015. Some of the many questions scientists have been able to help answer using...understood thanks to these technologies but many questions remain. The use of other technologies such as ...

...science - which can drive away even the most enthusiastic of learners.  What contributes to cognitive ...information and a logic to the types and timing of questions that are asked. Many things, therefore, will be...connection for your audience. I ask myself that question constantly (and so should you!)  Amount of information...as valuable feedback.  Listen. Think about the questions you’ve received, read the comments, seek feedback...  So now that you have all this knowledge, the question is: How do you know what’s too much (or too little...planned; other times students were fired up and enthusiastic and needed a faster presentation with more information...

...To LinkedIn or not to LinkedIn. That is the question. When presenting on building relationships (also...known as “networking”), one of the most common questions I receive is “Do I have to be on LinkedIn?” For...information available. When I am speaking at a new institution, for example, I will often see a whole bunch ...rule, but not that many). I’m asked a lot about requesting connections to complete strangers for informational...companies and organizations to see their news and job postings. 4) Use LinkedIn to pay it forward The best way... manager. This is considered intrusive and it’s still connecting with a stranger so is highly discouraged...

... gene expression. On the other hand, using a constitutive promoter in the same background resulted in ...over-represented compared to those further away. While constitutive promoters drove maximal gene expression near...during shifts in growth rate as compared to a constitutive promoter. To simulate the increase in plasmid...When they increased concentrations of IPTG, a constitutive promoter upstream of GFP increased expression...stabilized promoter while gene expression from the constitutive promoter did not. Similar levels of expression... al. placed the genes under the control of a constitutive promoter and then moved them into the genome...

... of interest in living cells. Challenges with existing RNA-protein interaction methods Not in a native...time point, but one minute of labeling with BASU still generated a >30-fold enrichment compared to E. coli...based proximity labeling methods, RaPID cannot distinguish between proteins that directly or indirectly ...sounds like a nifty technique, but what kinds of questions can it help answer? Ramanathan et al. outline ...of QKI decreased Zika viral RNA levels by 90%, suggesting that QKI is a host protein critical to Zika replication... study RNA-protein interactions. Do you have a question that RaPID could help answer? The RaPID plasmids...

...stranded RNA (ssRNA), while some exist as dsRNA. Still others use dsDNA to store their genetic information... made it into agriculture as species-specific pesticides and insecticides, an advancement over previous...sequence. All of the R-loops described above play distinct roles in promoting cellular processes and functions...While not perfect, these sensing systems work to distinguish between RNA of cellular vs. viral origin and ... each one acting on specific substrates and in distinct cell cycles. When RNA:DNA hybrids levels are allowed...appreciate double stranded isn’t just for DNA. Untwisting RNA is important for therapeutic delivery, viruses...

...participating in science and engineering. “Fix the Institutions” promotes gender equality in careers through...“Fix the Knowledge” or “gendered innovations” stimulates excellence in science and technology by integrating...study saved lives. Reduced use of estrogen plus progestin hormone therapy resulted in 76,000 fewer cases...the importance of analyzing sex, the National Institutes of Health (NIH) now requires that sex be included...Using cells from both sexes ensures that the diagnostics and therapeutic interventions you develop represent... work to be done! Researchers need to learn sophisticated methods of sex and gender analysis. Universities...

...regulation thats as sophisticated as a natural network. We could build a material as sophisticated as wood. To...Voigt explains it, his lab at the Massachusetts Institute of Technology has been “working on new experimental...to work with than regulatory proteins. There are still things we haven’t solved that stop it from being... carbons and oxygens. Nothing too much more sophisticated. There are also proteins, like antibodies. But...able to design new materials and chemicals as sophisticated as those that living cells and animals make....

...cycle once inside the host. While the virus can still enter (and thus infect) cells, this safety feature...Separation of retroviral packaging components onto distinct plasmids for safety.    Viral cargo-specific...cabinet and decontaminate after use. Virus-exposed plastics and other surfaces should be thoroughly decontaminated...decontaminated after use, typically with bleach (plastic) or ethanol (other surfaces). For AAVs, which are...should be added. After decontamination, single-use plastics and other handling tools should then be disposed... to reduce the risk of exposing yourself via needlestick or cut. Following all sharps procedures for your...

...Nanoblades to Existing CRISPR Delivery Methods When compared head-to-head with existing CRISPR delivery...editing efficiency and off-target effects compare to existing technologies, and their use for HDR editing and...Table 1). Additionally, hiPSCs and mouse BM cells still behaved like iPSCs and BM cells after Nanoblade ...pluripotency marker expression and response to LPS stimulation, respectively. Primary cell type Gene targeted...to difficult to target cells. When compared to existing Cas9 delivery methods, Nanoblades have just as...

...past, we've been working with the Rare Genomics Institute to provide plasmid grants to researchers working...mechanisms behind the immune defects in CDG patients are still poorly studied and understood.  In line with this...complexes, but a serendipitous discovery led them to investigate the use of a common laboratory chemical, guanidine...Engineering the human disease alleles into cells for testing in the lab will be greatly facilitated by CRISPR...sites in T4 lysozyme by deficiency-creating substitutions." Journal of molecular biology 277.2 (1998):...significance (VUSs) that are frequently found in genetic testing for syndromes that include developmental delay,...

...other species, but these studies generally leave questions about gene function wide open. What if two seemingly...over time? Undergraduates can help to answer this question! The Pathways Over Time project The Pathways ... a framework for these undergraduate research questions. The idea is straightforward. Instructors first...have been using yeast models (3, 4) to answer questions about the evolution of the genes involved in methionine...experiments that are used to answer our research question, the plasmids are also used to teach students ...and instructor resources is available at our Investigations in Molecular Cell Biology Website. Many thanks...

...An estimated 320,000 viruses can infect mammals. Even more abundant are the Earth’s estimated 1031 bacteriophages...instead to use the virus immediately upon harvesting. Interestingly, freeze-thawing host cells immediately... about the integrity of your virus, perform a diagnostic restriction enzyme digest and compare it to some...References 1. Anthony, Simon J., et al. "A strategy to estimate unknown viral diversity in mammals." MBio 4.5 ...

...deconstruct and understand this complexity using existing C. elegans mutants. For instance, we use a mutant...Minnesota has declared our makerspace an educational institution meaning that we can obtain known mutants at $7.00...makeup of the bacterial species in the gut of the domesticated housecat, and seeing how that makeup differs...cats with the goal of optimizing the health of domesticated cats and learning about feline microbiomes. ...pipeline. It’s possible that we’ll find something interesting and contribute it back to the kittybiome project...monitoring homemade microfluidic chips using the fantastic optics and fluidics platform it contains. Biotech...

...chance as entrepreneurs. To make things more interesting, we are working in a new technical discipline... cards, incorporation, laboratory lease, etc. I still flip to that page in my lab notebook from time to...worked weekends and evenings on the project, but still continued to generate new ideas as they popped into...doing makes sense. I can support that.” That was interesting. If people could be against GMOs as food but ... in the comments below. We’re happy to answer questions about this transition (so far) and what we’ve ...Biophysics and Biochemistry from the Illinois Institute of Technology in 2004, she accepted a commission...

...valuable laboratory tool are still an area of active research. New details are still being uncovered, and different...produce SuperFi-Cas9, a high-fidelity variant that still retains high cleavage efficiency (Bravo et al., ...et al., 2014). New discoveries on this topic are still being made! Conclusion Finally, we can summarize...their own advantages—and blind spots—to these questions. Each new structural insight also spurs the next...California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber...Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. Resources...

...The vast majority of bacteria are undomesticated which limits the tools scientists can use to study them...CRISPRi machinery into diverse bacteria species. Existing protocols can transfer CRISPRi into a single bacterial...transfer Mobile-CRISPRi offers two advantages over existing bacterial CRISPRi methods. First, its modular ...Knockdown efficiency, as measured by knockdown of a constitutely expressed RFP reporter, was variable across ...Klebsiella pneumoniae, and P. aeruginosa (all opportunistic pathogens) were grown in presence of trimethoprim...Elizabeth A., and Pamela A. Silver. "Harnessing undomesticated life." Nature microbiology 4.2 (2019): 212....

...researchers have engineered alternative Cas9s binding distinct PAM sequences. Now, Hu et al., working in David...so far! Why do we need PAM flexibility? IDT estimates that there is an NGG PAM every 42 bases in the...edits or knock-ins. Editing efficiency decreases drastically once your cut site is more than 10 bp away from...frequency. Despite the progress we’ve made, there is still a need for and interest in enzymes with shorter ...editing; for known promiscuous gRNAs, xCas9 3.7 still showed a 4.2-9.4-fold improvement in off-target:...these early data are exciting, there’s a lot we still don’t know about xCas9. “I’m not 100% sure xCas9...

...after graduate school, at least not while you’re still in the thick of it. About three or four years into...ways to get a taste of a variety of careers while still working towards your degree. For me, experimenting...The article submitted was hilarious, full of interesting information, and received a lot of attention ... said, “Boy, I love science,” and I realized, I still do too! Beyond the outreach experience I gained ...back into lab the next day and remembered why I still love science. Keep building on your time in graduate...look back at a positive, fulfilling experience. I still use my network and graduate school resources daily...

...? Depositing lab: The name of the Principal Investigator (PI) in whose lab the construct was made. The...The depositor name links to a page listing all of all this PI’s plasmids that are available through Addgene... plasmid page.  The paper link leads to a page listing all the plasmids from this paper that are available...Recipient Scientist Acknowledgement (RSA) asks requesting scientists to cite the depositing lab whenever...about how the vector was assembled, and enables requesting labs to utilize and modify the plasmid in their...will be to your colleagues. If you ever have any questions about our plasmid pages, please don't hesitate...