... 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...

... acid (DAP), a component of the cell wall. Once in the recipient cell, the CRISPRi DNA (purple) flanked... is propagated during normal DNA replication and cell division. Mobile-CRISPRi does not disrupt gene function...mobilize these plasmids for transfer to recipient cells during a tri-parental mating scheme. Mobile-CRISPRi...bacteria associated with human disease (S. aureus, Klebsiella pneumoniae, and P. aeruginosa) in addition to...antibiotic trimethoprim. When Enterobacter aerogenes, Klebsiella pneumoniae, and P. aeruginosa (all opportunistic...

...AmpliCan We chose to adapt Crispresso by Luca Pinello and Kendell Clement for batch analysis because of its...Leonetti Ryan Leenay Jason Li Max Haeussler Luca Pinello Kendell Clement Andy May Additional resources on the...members have taken up to a month to design a single 96-well plate for HDR that satisfy all requirements. To ...all the standard information that goes into a 96-well plate and comes out of it. Biotech companies are...

...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...

... 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....

...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...

... 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...

...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...

...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...

...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 ...

...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 ...

...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 ...

...plasmids to upregulate their expression in specific cells. With a more concrete understanding of the molecular...molecules targeting SMN1 and its partners in mouse cell lines. Dr. Di Schiavi hopes that these studies will... Mesothelioma  Noushin Nabavi is a Postdoctoral Fellow at the Vancouver Prostate Center and is affiliated...that affects the lining of the lungs is relatively well known, there are a variety of lesser known mesothelioma... of ~30 patient derived peritoneal mesothelioma cells lines and assess their tumorigenesis. These experiments...

...statistical tests – when to use them, what they can tell you, and how to identify which one is right for ..., and mode give you a summary snapshot without telling you anything about outliers or data spread within...Distribution - spread of data; typically, normal (bell curve) or skewed (see Fig. 2). Assumptions – ... up to two independent variables. This test will tell you if there are significant differences between... the means of your groups. However, it will only tell you if differences exist somewhere within your data...crop production (though you would not be able to tell if the dependent variables worked synergistically...

...the fall colors are inspiring you to paint your cells in equally vivid colors, you'll want to check out...AAV9-X1.1 enables expression in brain endothelial cells via an injection into the systemic circulation. ...levels compared to AAV9. You can now target these cells with AAV-CAG-GFP packaged in AAV9-X1.1!  We also...recombinase or a fluorescent marker in the various cell types of the basal ganglia. They work via newly-...before. We have a bunch of other new AAV vectors as well — including next-generation biosensors for GABA,...

...two-photon microscopy. Cell, 18, 3408 - 3425.e29. (2022). https://doi.org/10.1016/j.cell.2022.07.013.   ...voltages. This includes monitoring of fast bursts, as well as slow up/down voltage states in deep tissue, in...voltage fluctuations. Image credit: Liu Z, et al. Cell. 2022.  Find JEDI-2p viral preps here (and here...specific for human c-myc proto-oncogene product. Mol Cell Biol. Dec;5(12):3610-6.(1985). doi: 10.1128/mcb.5.12.3610...

...profit motive. We build this trust by keeping excellent records and maintaining confidentiality where ... Addgenies like the nonprofit company culture as well. We work every day to do a better job of enabling...Jessica, Joel, Jonathan, Justine, Karen, Kate, Kayla, Kelly, Kory, Kris, Leila, Lianna, Luke, Marcy, Mary, Max...Melina, Meron, Michael Lo., Michael Le., Michel, Michelle, Morgan, Natashia, Nicole, Olivia, Pamela, Racheal...Addgene Blog Learn about Our Collaboration with Fellow Nonprofit Seeding Labs Addgene Supports Rare Disease...

...circuits are generally linked to dopamine (DA), a well-known reward chemical and neurotransmitter. But ...firing patterns (or the frequency of its release) are well understood, the direct effect of DA binding to a...the brain has been in vivo microdialysis of extracellular fluid (Darvesh et al., 2011). This technique... across the semipermeable membrane from the extracellular fluid into the perfused liquid. The probe then...detection of dopamine in flies, fish, and mice." Cell 174.2 (2018): 481-496.PubMed PMID: 30007419. PubMed...

... remember what happened when drug X was added to cell line Y, or the phenotype of a mouse with a specific...comparison of the same experiment from different cell lines/model systems ReFigure allows you to make...the right experiment! The ReFigure extension also tells readers if a paper they’re reading has been linked... to show that a previously used reagent performs well, or you might publish a replication experiment that...immuno-oncology. Funding agencies such as the NIH and Wellcome Trust now accept interim research products (pre-registration...

...sweat, and tears, as well as the whole point of the paper. They should be able to tell your story for you...I am writing isn’t well written, I must write everyday in order to get better. Well what does that mean...interaction of critiquing others’ manuscripts as well as being critiqued added an extra level of application...

...allows you to sort for GFP-positive cells and to enrich for those cells that were positively transfected....introduction of a mutation into the genome, how can cells with that mutation be selected/screened? A4: Before...can use a selection marker to select transfected cells (for example, plasmid with a puromycin resistance...paper). If you detect positive HR, isolate single-cell colonies, grow them up, then perform individual ...