...gate which selects anything to the right of it, marking it as GFP+. Quantification for such graphs could...plot! The dreaded smear - fear not! When you are looking to study the level of protein expression, instead...Figure 2). These smears tend to occur when you are looking at proteins that are expressed regularly in cells...percentage positive or negative in your dot plot, looking at a histogram vs antibody plot would be better...

... the experiment in my head, double-checking details, or just taking a moment to relax. During really stressful...worth doing, and doing well, so make sure you’re taking time to do them carefully and thoroughly. Have ...however, many ways for you to reduce the chances of making an error. Try a variety of things, including (and...

...and molecular biologists use to tinker with DNA. Making changes to a DNA sequence can help scientists identify...components: the error prone PolI3M that’s fused to a nicking Cas9 (nCas9). Similar to mutagenesis with error...error-prone polymerase (PolI) that is fused to a nicking Cas9. A guide directs Cas9 to a site of interest...changes promote nCas9’s dissociation from DNA after nicking and yielded an enhance nCas9 (enCas9) EvolvR with...

... with translatable skills: working with data, working with people, working with complex processes Arm ...Ginkgo Bioworks, our software development team was working on lab operations software for next-gen sequencing...

...work best for your scientific question? If you’re thinking, ‘wait, those terms mean different things?!’, ... to clearly define what type of factor you are looking for and what process or outcome that factor functions... advantage – that is, you are not specifically looking at cells that can survive. Instead, all manipulations... screens are similar to CRISPR, but instead of knocking out the genes of interest (or the whole genome...

...question more and more students are asking. Teachers are also asking, “How do I engage my students in real-world...outcomes by emphasizing the importance of critical thinking, persistence, teamwork, and communication. And... the only informal synthetic biology club of its kind, running from start to finish during the academic...

...Ku then acts as a scaffold for recruitment of a kinase (DNA-PKcs) and a two subunit DNA ligase (XRCC4-...heterogeneous when constrained by sequence identities in flanking sequence (“microhomologies”). NHEJ doesn’t obligatorily...Renouf B, Renaud J-B, Sallmyr A, Ruis B, Oh S, Tomkinson AE, Hendrickson EA, Giovannangeli C, Jasin M, ...Inoue A, Matoba S, Zhang Y, Zhang F (2013) Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing...

...Developmental Editor at F1000Research.  With many changes taking place in the world of science publishing, and an...include in my paper? More and more journals are asking for detailed information on lab resources, such...enough information to replicate your experiments, making research more reproducible and your results more...journals as possible at the same time. Take your time picking the appropriate journal and explain in your cover...

...note: in this table, reprogramming efficiencies ranking as low, medium or high is based on the reprogramming... pluripotency of mouse cells typically involves making chimeras by injecting iPSCs into blastocysts and... injecting iPSCs into immunodeficient mice and looking for tumors that contains cells from all three germ...of iPSCs is the number one priority, with safety taking lower priority. Genomic integration is less of ... and Juan Carlos Izpisúa Belmonte. "Methods for making induced pluripotent stem cells: reprogramming à...

...autophagosomal membranes, so tagging it with GFP allows for tracking the localization or accumulation of pre-autophagosomal...an autophagy-independent manner. Additionally, tracking Atg8p or LC3 doesn’t provide information about...easier to study the mechanisms behind autophagy by tracking what’s being transported to the yeast vacuole ...vacuole (i.e. cytosol, mitochondria), rather than tracking generic autophagy markers. It’s particularly useful...

...like cross-linking immunoprecipitation (CLIP) identify RNA-protein interactions by cross-linking RNAs and...enrichment compared to E. coli’s BirA*. Faster labeling kinetics also means that BASU could potentially be used...RaPID RaPID sounds like a nifty technique, but what kinds of questions can it help answer? Ramanathan et al...

...promote tumor durability instead of repressing them, making it even harder to cure cancer. To understand how...factor, vascular endothelial growth factor, and interleukin-6 (Witkiewicz et al., 2009) which contribute ...metastasis of tumors through inciting inflammation. Speaking of inflammation, a noteworthy cell type in the...Witz, Isaac P. "The tumor microenvironment: the making of a paradigm." Cancer Microenvironment 2.1 (2009...

...with a heterozygous 4 bp deletion in MYBPC3 donated skin, blood and semen samples. Ma et al. designed plasmids...of only 27.9% and homology-directed repair (HDR) taking place in 41.2% of edited cells. The results were...CRISPR editing. Conclusions & implications One striking feature of this paper is the failure of the embryos...comprised of individuals with varied backgrounds. Talking about this study with non-biologist friends Like...

...the design of flanking overhangs. Although there are theoretically 256 distinct flanking sequences, sequences...their recognition sequence, creating four base flanking overhangs. Since these overhangs are not part ...clone up to 7 gRNAs into one destination vector, making multiplexing easy.  Disadvantages of Golden Gate...

...tweet in order to easily access it later. Pro tip Checking the number of retweets, likes, and comments on...recent papers that the scientific community is talking about and jumping into the discussion! Semantic...altmetric score, is also displayed in your feed. By clicking on a paper in your feed you can see suggested ...Career Research Toolbox blog posts on free tools for making scientific graphics post and social media for scientists...

...by starting with the least-abundant protein and working toward the most abundant. If the abundance of your...precipitates that permanently stain the membrane, making that section of the blot unusable for reprobing...ll want to check for the primary antibody by re-blocking your membrane and re-incubating with your secondary...is the one time in western blotting that you’re looking for a “blank” result! If some signal remains, you...

...localization in cells, or even single-particle tracking of the bound complex. There are so many FPs available...TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells... can even be bright enough for single-molecule tracking in live cells.  Controls  When designing experiments...editing (Feng Zhang Lab 2015)  Reference Feng, S., Sekine, S., Pessino, V. et al. Improved split fluorescent...

...another optimized platform. Gene Stacking As the name implies, gene stacking is the use of a combination of...engineering complex cellular behaviors requires making complex manipulations to cells. For example: in...reagents and strategies are not available to those working with yeast or plants. Using MoClo, or better yet...

...  Second, plasmids are easy to store and share, making recombinant antibodies the most practical choice... and producing antibodies from plasmids is much kinder to our animal friends! Finally, if we want better...improve genetically-encoded research tools, it is shocking that the plasmids and sequences for antibodies...10.1038/520295b Bradbury ARM, Trinklein ND, Thie H, Wilkinson IC, Tandon AK, Anderson S, Bladen CL, Jones B,...

...Epigenetic engineering with single-pot assembly Tracking cell-cell interactions New auxin inducible degron...Epigenetic engineering with single-pot assembly By Eric Perkins Though it’s easy enough to contemplate DNA in terms... own engineering plasmid tools in about a week. Taking advantage of Golden Gate cloning, the Haynes lab...human body and G-baToN provides versibility in tracking and modulating the interactions between cells....

... one place... well, lucky for you one is in the making by Dr. Zhuhao Wu and his colleagues! Dr. Wu and...for low-cost, high-quality antibodies for IHC. Working with collaborators and support from the Brain Research...enable scientists to have an intuitive guide for checking their staining and neuroanatomy without the fear...by submitting data, sending antibodies, and/or working with Dr. Wu please contact us here! Ava Shipman...

...This disease is caused by mutations in the common cytokine receptor gamma chain (c) and is characterized ...deficiency, Cystic fibrosis, Muscular Dystrophy, Parkinson’s disease, and HIV. A notable landmark study showed...AAV-based gene therapies are promising, scientists are working to overcome key challenges to their efficacy:  ...AAV vectors used to treat them. Scientists are working to overcome this issue by developing new capsids...

...Terskikh et al.) and even displays predictable kinetics over 14 hours of expression in C. elegans embryos...Miyatsuka et al.) Distinguish between models for trafficking to the lysosome by fusing Medium-FT to LAMP-2A...structure and a particular biological event (trafficking to a subcellular location, start of gene expression...change color from blue to red report on cellular trafficking." Nature chemical biology 5.2 (2009): 118-126...

...the development of a simple and scalable means of making targeted genetic edits. Cas9 is a DNA binding protein...Traditional Cas9 regulators In addition to its utility in making targeted modifications to DNA, Cas9 can be reprogrammed...potent Cas9-based transcriptional regulator without making any functional changes to Cas9. For instance, when...PMC4747789. 6. Platt, Randall J., et al. "CRISPR-Cas9 knockin mice for genome editing and cancer modeling." Cell...

...Hi-C (Han et al., 2018). Think of 3C methods as taking a snapshot of chromatin looping with the photo ...CAPTURE method is similar to other 3C methods: crosslinking captures chromatin interactions, a restriction...together to create loops of DNA. This is key to linking pieces of DNA that are far away from each other...Zhang, Y., Liu, Y., Liu, N., Botten, G.A., Cao, H., Orkin, S.H., Zhang, M.Q., & Xu, J. (2020). Multiplexed...

...The ocean is full of striking visuals from fluorescent jellyfish, bioluminescent phytoplankton, and colorful... see if your cloning experiment worked just by looking at the cells on the plate. For example, if your... identification is a good place to start before taking on more time consuming and expensive methods. While...Learn more about FRET Find protocols and tips for making plasmid constructs Resources on Addgene.org Find...

...Whether you’re new to in-house virus production or looking to improve your current methods, understanding ... add an additional viral passage. This involves taking your first round of virus, called P1 (or sometimes... and may need to be optimized for your purpose. Making your decision The final decision of which system...10430340260355347. Vaughn, J. L., Goodwin, R. H., Tompkins, G. J., & McCawley, P. (1977). The establishment...

...enter industry since I was drawn to the idea of working as a team to develop therapies that could directly... since you can’t afford to spend too much time working on something extremely risky or exploratory. Getting...it does not divulge any propriety information. Working as a team: Industry is all about teamwork. A single...environment, surrounded by lab members who are all working on related topics, scientific discussion is often...

...editing!  CRISPR functions in use But exactly what kind of editing are people doing with all these CRISPR... modifier. We used these categories to see what kinds of CRISPR tools people were depositing and requesting... share useful tools, and as such, we’re always looking to understand our community better. We therefore...year, or approximately 104 plasmids shipped per working day, including appropriate MTAs and customs forms...

... unknown, it is entirely possible that you are working with a low copy plasmid. When increasing the culture..., or a home-brew optimized for plasmid yields. Spiking your media with supplements such as magnesium salts...can optimize your growth time, temperature, and shaking speed to maximize cell density and plasmid yield...especially those in deep-well plates, require a faster shaking speed for proper aeration (260-300 RPM). 37℃ doesn...

...Institute and Harvard Medical School. Scientists making transgenic organisms with Cas9 should be aware ...should be conducted with extreme caution. Why does making transgenic organisms with Cas9 risk creating a ...and single guide RNAs (sgRNAs) with appropriate flanking homology into a germline cell can create an RNA-guided...mosquito species. Reproductive confinement involves working with laboratory organisms that can't reproduce ...

...accurate, as these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond...cleavage efficiency. How do I get started? If you are looking to co-express your gene of interest along with ...with a plasmid that has multiple cloning sites flanking the multicistronic element(s), or you could replace...within the same multicistronic vector, effectively making a construct that expresses multiple unique genes...

...11326284. 4. Bennett J, 2017. Taking Stock of Retinal Gene Therapy: Looking Back and Moving Forward. Molecular...pioneer in the field, Dr. Jean Bennett started working on ways to deliver genes to correct genetic conditions.... Jean Bennett (UPenn) that resulted in the groundbreaking 2001 study showing that AAV-mediated delivery...

..., are right around 250 nm, making it difficult to study the inner workings of the synapse by conventional...caused by loss of protein during digestion, or masking of epitopes during sample anchoring and gel polymerization...et al. 2019). Perhaps it’s the tool you’ve been looking for too!     References and resources References...

...appealing, but also scary. I do a lot of public speaking where I enjoy communicating face to face with ...early and brush up on best practices for LinkedIn networking. I was an early adopter of the LinkedIn blog ...ve said this before, but I also highly recommend taking every opportunity you get to speak publically about...Share Your Science 6 Tools & Tips: Online Social Networking for Scientists Sharing Your Lab Protocols: Using...

...each sample by clicking on the sample name or on its corresponding bar graph entry. Clicking to initiate ...engineering has revolutionized the gene editing field by making experimental workflows considerably easier, faster... download the entire analysis as a ZIP file by clicking the Download Analysis Data button on the bottom...

...collecting fibroblasts, which require a more invasive skin biopsy procedure. This becomes a major benefit when...patients, specifically children and patients with skin disorders for research and clinical trials, as it...usually require about 5-10 passages. Furthermore, working with Sendai virus requires stringent biosafety ...Road Ahead." Journal of Alzheimer's disease & Parkinsonism 6.5 (2016). PubMed PMID: 27882265. PubMed Central...

...replication by binding to the origin of replication and blocking replication machinery (del Solar, 1998). As the... on each plasmid and pair the plasmids together (akin to sister chromatids coming together in eukaryotic...centromere-like region, and the NTPases end up “walking” the same plasmids to one daughter cell, instead...occurs for several reasons, including one plasmid blocking replication of another and one plasmid outcompeting...

...as well as high spatial and temporal resolution, making it ideal for visualizing gene expression in vivo...ultrasound contrast. We (the authors) are currently working on improving and optimizing ARGs, with Marjorie...They also did not scatter ultrasound nonlinearly, making them difficult to distinguish from background tissue...intraperitoneal doxycycline injections to enable their tracking with ultrasound imaging (Fig. 2d). Creating a ...

...process was working as expected, we moved forward with creating the next process and linking the output...needs of the user. Thus, a key criteria we were looking for was the ability to re-use or borrow existing... end of the script or by opening the files. By working on one process at a time, and testing a small set...

...such as cell movement/motility, development, cytokinesis, and muscle contractions (think skeletal muscle...contexts within the fly including oogenesis and cytokinesis. Why name this protein after a vegetable substitute...encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila." Cell 65.7 (1991): 1177-1189. ...resources on the Addgene blog Read our Quick Guide to Working with Drosophila Read our Plasmids 101 blog series...

...stem cell research initiative at the King Abdulaziz University, Kingdom of Saudi Arabia. Bioinformation, ...markers are read in parallel.  Let’s imagine we’re looking for cells in a sample that are negative for marker...controls. You can usually tell where this point is by looking at the different populations. In the case of Figure...

...situation differently, some adopting simple note-taking tools, others using excel sheets or organizing ... on the market today, ranging from general note taking tools, to more scientific field-based solutions...of use of the software. We identified that when talking about ELN costs and limited budgets, “a large percentage...ELN will support that. In addition, if you are working for a large institution or company, site licenses...

...forever!  This can be an advantage for researchers looking to generate long-lasting changes in cells. However...inserting viral DNA into the host genome. Without a working integrase, IDLVs can still deliver their genetic...Considerations for IDLV use If you’re reading this and thinking “Gee, I’d love to give these a try myself,” look...Linch, D. C., Bayford, J., Brown, L., Quaye, M., Kinnon, C., Ancliff, P., Webb, D. K., Schmidt, M., von...

...experiment. What is immunofluorescence? Generally speaking, IF is an application in which antibodies that... different approaches to be familiar with when talking about IF, direct and indirect. For direct IF, researchers...different colors. Multiplex immunofluorescence Speaking of different colors, having a palette of secondary...permeabilization, antigen retrieval, and sectioning. Blocking Block is used to reduce non-specific binding...

...Iannaccone et al., 2009). Rats are larger than mice making them more useful for certain studies that involve...genetics and developmental patterns are conserved, making them great models for human disease research. One...eggs are large, opaque, and produced in abundance, making them useful for in vivo studies. These attributes...flowers. Image from BlueRidgeKitties. If you are working with Arabidopsis check out the GreenGate Toolkit...

...fit a big gene into a tiny vector like AAV? By breaking the transgene into smaller pieces. Reassembly ...following transduction.  So what’s going on here? The working model is that the oversized AAV genomes are fragmented...Surveillance Mechanism That Eliminates Transcripts Lacking Termination Codons. Science 295:2258–2261 . https... Hirsch ML, Li C, Bellon I, Yin C, Chavala S, Pryadkina M, Richard I, Samulski RJ (2013) Oversized AAV...

...Brainbow labeling has also been applied to cell marking and lineage tracing in other systems, such as zebrafish...directly trafficked to cell membranes. Membrane trafficking of farnesylated derivatives enables labeling ...fluorescence due to the addition of translation-blocking STOP cassette immediately following the promoter...dev.094631. PubMed. Multiplex cell and lineage tracking with combinatorial labels. Loulier K, Barry R,...

...this post, we didn’t come across a broadly used working definition of systemic capsids. After some consideration...Beadle, K., Jang, M.J., Kim, H.M., Rajendran, P.S., Tompkins, J.D., Shivkumar, K., Deverman, B.E., Gradinaru...Chen, X., Wolfe, D. A., Bindu, D. S., Zhang, M., Taskin, N., Goertsen, D., Shay, T. F., Sullivan, E. E.... I. G., Pacouret, S., Chen, A. T., Chan, Y. A., Ryvkin, D., Gu, C., & Deverman, B. E. (2022). A high-efficiency...C., Lagerborg, K. A., Tellez, L., Krunnfusz, A., King, E. M., Ye, S., Solomon, I. H., Tabebordbar, M.,... Tabebordbar, M., Lagerborg, K. A., Stanton, A., King, E. M., Ye, S., Tellez, L., Krunnfusz, A., Tavakoli...

... channel with fast kinetics may be required, whereas a channel with slower kinetics can be used for lower...each light source used.   Channel and activation kinetics Some channels turn on and off very rapidly in ...thorough characterization data, like spectral and kinetic activation information, in the supplemental data...