...there isn’t a template strand left behind that the cell can use to repair. Figure 3: General overview...Occasional cleaving of the target strand is not as well understood but likely occurs in similar regions....complex in nucleotide unwinding steps. Molecular Cell, 70(3), 385-394.e3. https://doi.org/10.1016/j.molcel...formation by a type I-C CRISPR Cascade. Molecular Cell, 83(5), 746-758.e5. https://doi.org/10.1016/j.molcel...broad and unidirectional genome editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038...CRISPR–Cas systems for genome engineering in human cells. Nature Biotechnology, 37(12), 1471–1477. https:...

... make it difficult to properly gate your desired cell population. By compensating between fluorescent ...online manual for your cytometer should be able to tell you the laser configuration. In general, if two ...Figure 1A). Applying compensation means you are telling your cytometer to adjust the fluorescent signals... your fluorescent markers are staining for. If a cell is positive for both markers, it will show up as...Figure 3: Flow plot for a double positive sample. The cell population in the top right corner of the plot is...learning! Ashlyn has a research background in CD8+ T cells and cancer immunotherapies. She is currently a research...

...different kinds of information on our plasmid pages? Well, to help make the most of these pages, we've written...Addgene’s plasmids is an indication of popularity. A yellow flame indicates that a plasmid has been ordered...process and these can be found in this location as well. These maps make it easy for scientists to see if...analyzed using Addgene’s Analyze Sequence tool. Excellent, how can I check if the plasmid will be within...resistance, and optimal growth temperature are listed, as well as the name of the strain.  Most Addgene plasmids...such as some of our viral vectors, in NEB Stable cells. Resource information: This section contains information...

...favorite parts for plant synthetic biology Addgene: Tell me about the Golden Gate Modular Cloning (MoClo)...really easy to clone genes for expression in plant cells. Sylvester and I chose the 96 most useful things...used as we have tested them to show that they work well in plants and give similar stability to transcripts... parts available in your lab - if you get the 96-well kit - everything is so easy. We have undergrads ...a real person, so that students see academics as well-rounded human beings. It’s also a great communication... that want to have a big impact on the world and tell them to work in plant science. I think their chances...

...CRISPR complex much easier in various cells, including primary mouse cells. We believe that this system will...In 2014, you joined Addgene's Advisory Board, as well as the Editorial Board of Scientific Reports and...US, I very much enjoyed interactions with many excellent colleagues at NYU and visiting scientists, such...In addition, I've been very lucky to work with excellent colleagues in my lab. On the other hand, I feel...good for Japanese science. I really miss the intellectual environment in the US in this regard. In addition...

...occurs when bacterial cells regulate gene expression in response to changes in cell density. At the mixer...cohort of friends and colleagues had moved on as well, so I knew very few people. I didn’t really fit ...Singletons gained from spending time with a mentor, as well as having a cohort experiencing similar situations...introduced to several people by their mentor, as well as their bin-mates.  They liked us. They really ...Hendrickson, Jonathan Jacobs, Mo Kaze, Christina Kellogg, Heather Martinez, Pat Schloss, Tara Smith, Marc...

... it, and many of my scientist colleagues have as well. Making the transition from the bench can be jarring...scientist going through even the worst experimental dry spells. Losing that sense of discovery can be hard since...place. The feeling of camaraderie one gets from a well-functioning lab team. Obviously not everyone gets...applying. I probably did not respond to her question as well then as I would now. I would have to repress laughter...the right set of tools to help you do your job as well as possible. It’s in their best interest to do so...the newer members of our Scientist team says, “a well-run meeting where everyone has a voice and the group...

...choose a molecular cloning technique that will work well with a given set of resources, time, and experimental...the next step is to transform competent E. coli cells and select the positive clones. The entry clone ... expression of the human KRAS gene in mammalian cells. STEP 1: Generate an Entry Clone There are a few...ccdB gene. Since the ccdB product is toxic to the cell, Gateway cloning efficiency can reach >99%. ...digest! Then, you can transform or transfect the cells that you want to use for your experiments and verify...Phage (Lambda) and Higher Organisms (Cambridge, MA: Cell Press). Additional Resources on the Addgene Blog...

... edit A → Y (Y = T or C) in 67.17% of screening cells (Tong, Wang, et al., 2023). Based on this promising...the base editor. Black arrows indicate natural cellular processes. Created with BioRender.com. The...Cas9 nicks the non-edited strand to encourage the cell to repair the DNA using the edited strand as a template... efficient A•T-to-C•G base editing in mammalian cells and embryos. Nature Biotechnology, 42(4), 638–650...programmable T-to-G and T-to-C base editing. Molecular Cell, 84(7), 1257-1270.e6. https://doi.org/10.1016/j....efficient T-to-G and C-to-G editing in mammalian cells. Nature Biotechnology, 42(10), 1538–1547. https:...

...(or even in the middle of proteins) and performs well in many different antibody-based applications.  ...Randall, R. E., Young, D. F., Goswami, K. K., & Russell, W. C. (1987). Isolation and characterization of...useful addition to the toolkit for visualizing cellular processes in C. elegans: mScarlet3 for applications...biologically relevant forces at focal adhesions in living cells in several different contexts.   Figure ...substrate furimazine. Right: Demonstration of PILATeS. Cell adhesion to a PILATeS-functionalized coverslip creates...minutes.  Find PILATeS plasmids here! Zhong, B. L., Elliot, J. M., Wang, P., Li, H., Hall, R. N., Wang, B....

...used to deliver Cas9 and other genes to mammalian cells. For this reason, the top requested plasmid of 2017... coat the virus and help determine the types of cells that it can infect. The VSV-G envelope is widely... lentiviruses to infect many different types of cells. This means researchers who use VSV-G can deliver...deliver their genes of interest to a variety of cells and study their effects. To date, nearly 900 articles...in studies of the secretome, ubiquitin ligase, cellular senescence, autophagy, CRISPR, and so much more...

...fluorescent proteins as scaffolds for cell-specific gene manipulation.” Cell 154(4) (2013): 928-939. PubMed ...Cepko Interested in Working on the Eye 9:05-10:16 : Cell Targeting in Retinal Gene Therapies 10:17-14:34 ...28:39 : Methods for Delivering Multiple Genes to Cells for Gene Therapy 28:40-35:10 : GFP Activatable Tools...publication at Addgene. 2. Tang, Jonathan C.Y., et al. “Cell type-specific manipulation with GFP-dependent Cre...

...other people, but resolving your own conflicts as well. What I really brought to the table was empathy,...you can identify them in yourself and learn how to sell them. Types of Conflict Before explaining my approach...occurs when people or teams who normally get along well with each other disagree about a specific decision...good listener absorbs and remembers and makes the teller feel heard. I am not always a stupendous listener...will accept. If you are able to broker a compromise well, you can help everyone walk away from a conflict...solution C. And if a conflict resolver does their job well, not only will solution C be better than either ...

...double-strand break (DSB). DSBs can be caused by intracellular factors such as nucleases and reactive oxygen...repair fix DNA double-strand breaks?  In eukaryotic cells, repair of DSBs occurs primarily by two pathways...used, however, these are generally more toxic to cells than their plasmid counterparts. Another consideration...how to fix a broken relationship. Pardo B, et al. Cell Mol Life Sci (2009). PubMed. Regulation of DNA...break repair pathway choice. Shrivastav M, et al Cell Research (2008). PubMed. Mechanisms of DNA double-strand...Improve HDR Efficiency for CRISPR Editing in Human Cells Resources on Addgene.org Browse All CRISPR Plasmids...

...giving science talks and how to make them serve you well. The happy byproduct might just be a career opportunity...Listen to their stories and narratives (see below “Tell a story”) and note how they build suspense. Notice...host told me and wished I had been more careful. 3. Tell a story We are biologically programmed to listen...video for a job talk, but if you can get it working well, everybody loves a great animation. 8. Predict questions...good topic or story is all you need, if you do it well.  Resist the urge to show all or even a lot of data... I hate it when I watch myself speak, but others tell me that I keep the talk moving enough that they ...

...degraded, cell still can methylate DNA   hsdS(rk-,mk-) Unmethylated DNA not degraded, cell cannot methylate... gal Mutation in galactose metabolism pathway Cells cannot grow on galactose only gyrA, gyrA96 DNA...permease inactivated, lactose cannot be taken up by cell mcrA, mcrBC Inactivation of pathway that cleaves...gene that maintains the plasmids at ~250 copies per cell.  F- ∆lac169 rpoS(Am) robA1 creC510 hsdR514 endA...rpsL20 xyl-5 leu mtl-1 Stbl4   Electrocompetent cells for cloning and storage of unstable DNA and lentiviral...

...institutions in Ghana and most recently got his PhD in cell and molecular biology at the University of Edinburgh...developing novel systems for bioconversion of cellulosic biomass to useful products under Professor Christopher...Plasmid Grant. For this grant, we’re working with fellow nonprofit, Seeding Labs, to distribute plasmids...Kwabena plans to use his research experiences as well as plasmids from Addgene to bring new opportunities...biology, DNA assembly methods, and engineering cellulose degradation. 18:21 - 23:26 : Kwabena's plans for...

...transferable skills while you are also learning to be an excellent scientist. Leadership and management Take a lead...always involved. Demonstrating your ability to work well with others can be done in many ways… and many labs...supportive editors so your first drafts are always excellent Technical knowledge and creativity Become a “...Employers and academic departments want to be able to tell that you know about something and are passionate...local networking and trade organizations Another excellent perspective on this topic can be found on the ...

... vectors and genetically target them to specific cell populations. They demonstrated use of the LITEs ...non-invasively, its use is not restricted to cultured cells but extends to freely moving animals, as established...novel mode of optogenetic control of endogenous cellular processes." The LITE system can now be applied...no means limited to particular model organisms or cell types. “This tool is really flexible and modular...supernatant. (The details can be found here.) So tell us, readers, how will you get creative with LITE...

...immunocytochemistry (ICC) when the sample is cultured cells or immunohistochemistry (IHC) with tissues), where...know if they co-localize at the same place in the cell? Or you know that the presence of several biomarkers...tissue sections, methanol- or paraformaldehyde-fixed cell samples, or other preparations. Sequential IF For...anti-CASPR (green), and anti-Kv2.1 (cyan). (B) Cerebellum labeled with anti-GABA-AR β1 (white) and anti-GABA-AR...hippocampus, or counting the number of puncta in each cell. When making figures for a paper or presentation...approach for deep phenotyping and spatial analysis of cells in complex tissues. Proceedings of the National ...