...fluorescent protein: identification of the molecular determinants of color and applications in fluorescence... be used as a fusion tag for a wide range of applications from traditional imaging to FRET and stochastic...492 nm, respectively), they gave this a teal classification rather than cyan and named the protein mTFP1...the fusion protein localized to their expected locations. An analysis of mWasabi gave similar results. ...fluorescent protein: structural characterization and applications in fluorescence imaging." Biochemical Journal...

...off-target sites across the genome and pinpoint the location of mismatches based on the sequences of the genome... Figure 1: Schematic representations of the indicated unbiased genotyping options. Figure reproduced...those relating to DNA structure (e.g. epigenetic modification, bulges) that may also impact off-target edits...cell-based methods are genome-wide, unbiased identification of double-strand breaks (DSBs) evaluated by...Genotyping Options Technique Detection Limit Application Advantages Disadvantages GUIDE-Seq 0.1% Cell-based... HTGTS Not reported Cell-based Identifies translocations Limited by chromatin configuration, produces...detection of KRAS mutations: diagnostic and clinical implications." The Journal of Molecular Diagnostics 12.4 ...

...degradation, blocking translation, or chromatin modifications of complementary gene sequences (Figure 1). ...silencing can be easily induced by exogenous application of long or short dsRNA. However, it is important...unmodified dsRNA elicits immunogenic reactions that complicate the effectiveness and interpretation of RNAi....have benefited from the ease of use in dsRNA applications. Figure 1: Schematic of an RNAi-inducing...between targets) or multiple target sequences are concatenated. hpRNA has been extensively used in plant research...selection reporter whereby survival itself is indicative of active gene silencing. We achieved this by...Internal Marker for Silencing Allows for Rapid Identification of Loss of Function Phenotypes. Plant Biology...

...genetic code of living organisms and its societal implications even before the discovery of genes themselves...explores the concepts of animal testing and modifications. The premise of the novel involves Dr. Moreau...2018) were highly successful in the box office, indicating the insatiable appetite of the moviegoers for...induce a double-strand break at the target genomic location. This is typically followed by non-homologous ...point mutations in the genome. They fused a catalytically impaired Cas9 protein to a DNA-editing enzyme...deaminase to introduce a C→T change at precise locations in the genome. This was soon followed by the adenine...us to always consider the ethics and broader implications of our research.  Want the Genome Editing Trading...

...equipment sharing or a collaboration – it requires re-location of the scientist to the new institute where their...equipment! Preparing for your visit   Design and communicate a research plan Your time at the new research... and you are set up for success. If you are communicating with a professor or institute, ask if you will...out to your bench mentor (if you have one) to communicate your plan as well. Making logistical and funding...expect to generate data that will contribute to a publication? A grant? A patent? It is never too early to ...Resources Resources on the Addgene blog Fostering communication in remote collaborations Transferable Skills...

...unique barcodes for each TF help streamline identification. While the Zhang lab controlled their multiplicity...single TF was delivered to each cell, future applications could deliver multiple TFs to each cell, allowing...processes. For help with library transformation and amplification, refer to the Zhang lab’s previously published...published protocol and its relevant modifications on the pooled library webpage. Disease models Good disease...generate, in part because of difficulty with replicating the specific dysregulation in the disease. For...Addgene.org Plasmid Pooled Libraries Pooled library amplification Zhang Lab CRISPR Plasmids The Human TFome Library...

...the classical purification protocol involving iodixanol gradients. The current purification protocols for...particles accumulate inside the producer cells and purification protocols often describe a process that begins...lysis to release AAV particles with subsequent purification steps. It’s also possible for AAV vectors to... This is particularly useful for therapeutic applications in vivo where endogenous anti-AAV antibodies...Adrienn, et al. "Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous...

...fields and geographical locations. In addition to providing reagents and educational resources for scientists...formation. The quest for plant compounds to treat complications of diabetes Gloria Otito Izu, PhD Candidate...nephropathy, one of the major pathophysiological complications of diabetes mellitus. Recombinant human proteins... employing synthetic biology resources and microfabrication technologies to generate a self renewable ... in the river, take water samples at specific locations, measure fluorescent signals from the sensor, ...

...tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for ... Another example of reagents that have broad applications but have proven useful to the study of SARS-...vectors VSV-G and psPAX2, which were used in a publication by Xiuyuan Ou, et al. that characterized the ... requested these plasmids from Addgene. This publication then resulted in the development of a new plasmid...production and distribution is possible due to the dedicated facilities and manpower that a repository provides...

...Collaboration and creativity are pivotal in writing publications/grants too. As described by several works (Wuchty...backgrounds.” This work outlines the need for clear communication, fair credit through authorship, and acceptance...remotely. The authors stress the fact that “miscommunication, a lack of leadership, and inappropriate tools...approaches can lead to frustration, delay of publication, or even the termination of a project.” Take ...setting. Check out the blog post “Fostering Communication in Remote Collaborations” to get more ideas....Lewis ASL, Weyhenmeyer GA, O’Reilly CM, Lofton ME, Catalán N (2018) Ten simple rules for collaboratively writing...

..., Nature Communications, 2019. PubMed PMID: 31451697. Genetically encoded voltage indicators for use ... many aspects of ApoB-LP biology, including quantification of abundance, size, and localization in vivo...Pubmed PMID: 23068583 Thierer et al, Nature Communications 2019. Pubmed PMID: 31366908 Adapting sigma54... by Brook Pyhtila Genetically encoded voltage indicators (GEVIs) change fluorescence in real time when... excitation light intensities have made these indicators difficult to work with.  The Gottschalk lab has...

...Luciferases are a class of enzymes capable of catalyzing chemical reactions in living organisms resulting...gene, such as luciferase, usually serves as an indicator of transcription within cells, where detection...photon emission is measured resulting from the catalysis of a chemical reaction requiring luciferin, ATP...Background Lower Higher Post-translational Modification Not Required Required Photobleaching/Phototoxicity...blue light with a peak emission at 480nm upon catalyzing a chemical reaction with the substrate coelenterazine...compatible substrate must be provided for the enzyme to catalyze the light-producing reaction. Many luciferase ...

...These properties allow for efficient signal amplification, making biotin conjugated antibodies good options...especially if you start to consider clinical applications). But there are a few other classes that we ...proximity ligation, immuno-PCR, and single-cell applications (Hegazy, et al., 2020; Niemeyer, et al., 2007...also conjugate antibodies directly to beads for purification purposes.  Coupling up - antibody conjugation...added to antibodies post-translationally. 4) Modifications made to antibodies, such as inclusion of non-canonical...Detecting Protein-Protein Interactions and Protein Modifications in Cells and Tissues in Situ. Curr Protoc Cell...

...days prior to the implementation date, our data indicated that, while 69% of all postdocs were expected ...sent, with the lack of transparency and poor communication of their plans (many postdocs - and in some ...to reach out to institutions with a statement indicating that we encourage them to keep raising salaries... little correlation with cost of living at the location of the institution. Continuing efforts to gather...Director of Future of Research. He is interested in advocating for and with junior scientists, to help them ... Bring Increased Postdoc Wages Read about the Advocating for Science Symposium and Workshop Learn about...

...rounds of replication are initiated before the cell has divided. This means that genes located near the...problems. Rapidly dividing cells initiate genome replication more than once per cell division and thus, genes...genes closer to the origin of replication will be overrepresented compared to those further away. As a...maximal gene expression near genomic origins of replication, the stabilized promoters created by Segall-Shapiro...the control of IPTG. Because RepA is a plasmid replication initiator protein, they could alter copy number...

...detected with tyramide signal amplification. Tyramide signal amplification occurs when HRP converts a fluorescently...reporters (i.e. fluorescent dyes, HRP, etc. ) after purification. Antibodies are difficult for an individual ...culture media. Their His tags allow for further purification, if desired, but it's not required for their...some reporters are better suited for particular applications. Read on to learn more about each reporter or...Pros Cons Antigens HRP (P) Tyramide signal amplification of HRP Staining cultured cells and tissue sections...

...red fluorescent proteins) allow simultaneous identification of more than one protein of interest. However...problematic, because the DREADD receptors need to be located on the plasma membrane to be activated by the DREADD... species (bottom panel in Fig. 1). Our results indicate that the tag protein may have an important influence...fluorescence expressed in the neuropil conceals identification of opsin-expressing cells). For other things...Rodgers L, Wilson IA, Lerner RA, Wigler M (1988) Purification of a RAS-responsive adenylyl cyclase complex...(2013) Overview of Affinity Tags for Protein Purification. Current Protocols in Protein Science 73: . ...

...members of their pathways, but where the kinases are located, their signal strength, and how long their signals...and MEK1 is active or “on.” Dronpa’s color thus indicates MEK1’s current state (Green when off, dark when...domains were inserted at sites homologous to the locations they were attached to in MEK. The photoswitchable...kinases good for? There are a number of potential applications for photoswitchable kinases.  Below are a few...signaling by using psMEK1 and an ERK kinase translocation reporter with a mRuby2 fluorescent reporter ...endogenous ERK, and its localization serves as an indicator of MEK1 activity. When MEK1 is active, it phosphorylates...

...digital notebook and make sure you note the freezer location of every plasmid in your collection. Use one of...access and use the materials they need, and avoid duplicating your effort. In addition, if you should choose... important step in ensuring quality, as it will catch any errors made during the cloning, sample handling... for more tips on sequence analysis, plasmid verification, and using NCBI BLAST. Tips for high-throughput...result against the expected sequence for quick verification, which can be done through the align tab of ... plasmid verification...

...As evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR ...nickase variant, lowering Cas9 expression, and truncating gRNA sequences used for targeting; however, these...on-target and off-target activity at multiple genomic locations in HEK cells. Further testing of two of the mutants...PubMed PMID: 26628643. Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal...PubMed PMID: 26735016. Find plasmids from this publication at Addgene. 5. Chen, JS, et al. "Enhanced proofreading...PubMed PMID: 28931002. Find plasmids from this publication at Addgene. Resources on the Addgene Blog Read...

...repurpose popular GFP reporter lines for more sophisticated experimental manipulations, saving the time ..., 98% of TdTomato expressing cells were GFP+, indicating a robust, but specific system. T-DDOGs also successfully...systems are easily adaptable to neuroscience applications, including optogenetics, and they should make...Could Cas9-DOG be developed soon to allow genome modification only in a small subset of cells? Other proteins... PMCID: PMC4096992. Find plasmids from this publication at Addgene. 2. Tang, Jonathan C.Y., et al. “...PubMed PMID: 26258682. Find plasmids from this publication at Addgene. 3. Kirchhofer, A., et al. “Modulation...

...choose well. What are their qualifications? Do they have certifications? How can they demonstrate their...  Check out Joanne's Reddit AMA I am a strong advocate for peer mentoring, but volunteer mentors are ...help you. A good coach usually will be a clear communicator and make direct suggestions for improvement ...coaching is nice if you can get it.  Because I am located in Cambridge, MA (and because we have a lot of ...she is especially excellent in coaching for communication skills. Industry transition is also an area ...

..., its dimeric nature can interfere with some applications. Several labs have been working to produce monomeric... for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038...library generation and screening that led to identification and characterization of AAV.CAP-Mac. Image ...rodents and broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467...proven itself a good option for essentially all applications. Addgene’s plasmid collection includes over ...clone (M2) has been widely used in a variety of applications. Find anti-DYKDDDDK recombinant antibodies here...

...the best, or even which one will work for your application? Like many scientists, I have spent hours sifting...produced from a new set of animals), and wrong application (different experiments/conditions can change ...differently over time), compatible with the desired applications (such as immunohistochemistry (IHC) or western...colleagues! Dr. Wu and his lab members (like me!) are dedicated to creating a public and accessible database for...expectation and comparison on the antibody signal’s location. If a distinct imaged pattern does NOT align with...literature and is unrelated to the target protein, we categorize the antibody as having non-specific binding in...

... biological reagents have a ton of data on the catalog page. It’s not always super easy to navigate the...originally described in a peer-reviewed scientific publication. We love science, but we don’t have the resources...questions about an Abcam antibody. Have your catalog number ready. I've attempted to answer several ...Addgene support, make sure to mention the plasmid catalog number, or at least the plasmid name in your initial...be fairly fast, getting the full answer to a complicated question may take some time. If the customer ...message Answering customer service questions is a delicate balance of knowledge, patience, and diplomacy,...

...experiences to undergraduate students (1, 2), but educators face significant challenges in designing suitable... become very different due to a whole genome duplication and extensive rearrangements in the S. cerevisiae...overexpression vector. This plasmid contains origins of replication and selectable markers that allow students to...met deficiencies (manuscript in preparation), indicating that Met protein functions have been largely ... research into undergraduate courses and in communicating science to the public. You can find out more...). Vision and Change in Undergraduate Biology Education: A Call to Action, Washington, DC. 2. President's...

...scientific poster apart? First, a good poster should catch the audience’s eye and draw people in. That can ... sections should be as concise as possible, communicating only the most important results to your audience...have the advantage of being able to verbally communicate your work, so you don’t need every little detail...may choose to display it as rows. It can be more catchy because it is generally less common and will stand...advantage of being different and potentially more catchy, but it can be trickier to make figures fit nicely...relevant to the work you are presenting. The latter is catchy, but beware. A distracting background or clashing...

...chemical replication of RNA in the absence of evolved enzymatic machinery (i.e. RNA replication with no...Guangzhou and the Science City The conference was located in the modern, relatively new “Science City” area...and technology companies and start-ups choose to locate there. I made time to go into the city of Guangzhou...inheritance that might be less error prone than DNA replication. He too mentioned how “surprising” small RNAs...Steitz quipped that RNA in the ribosome is in a complicated conformation “not like DNA which has a boring...

...antiviral defense). Cascade is brought to the target location using a gRNA and can then recruit Cas3, which ... type I, the most abundant type in the Class 1 category. These type Is can then be further broken into...of DNA much like a fisherman would reel in a big catch. It was originally suspected that Cas3 moved a couple...Mashimo, T. (2022). Genome editing technology and applications with the type I CRISPR system. Gene and Genome...unidirectional genome editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-...Koonin, E. V. (2015). An updated evolutionary classification of CRISPR–Cas systems. Nature Reviews Microbiology...

...so most labs opt to purchase adenoviruses from dedicated adenoviral production labs or companies. Production...something you may have to optimize for your specific application. With mammalian systems, it is standard to do...more cells, while AAV can be titrated following purification using qPCR or digital droplet PCR. Insect cells...to collect your viral supernatant. Baculovirus replicates inside insect cells, and its lifecycle ends in...titer the virus, or go straight into your viral application, like protein production. Titration of baculovirus...Sf9 Time to collection 2–4 days 5–10 days Purification Ultracentrifugation (AAV); centrifugation (lenti...

...targeting anywhere in an exon. But things get more complicated if you’d like to use homology directed repair...They created a selection phage (SP) to carry catalytically dead SpCas9 (dCas9) fused to the bacterial polymerase...test xCas9 3.0-3.13, Hu et al. first restored catalytic residues D10 and H840. To ensure that the clones...loci displayed a broad range of PAM sequences, indicating they may be less predictable than SpCas9 off-... that we have only scratched the surface of modifications that can be made to Cas9. It is a pleasant surprise...editing rates. This PAM flexibility should expand applications that require very specific targeting, like homology...

...retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...wild-type viral particle — and of the normal, replicative life cycle — can help you understand these seemingly...of a non-living viral particle, and how we as dedicated researchers can exploit it. This blog was written...Hutcheson for her assistance with lentiviral replication.  Resources and References More resources on...101: Types of Viruses Viral Vectors 101: Viral Applications Resources on addgene.org Addgene's Lentiviral...acid of the Bryan strain of Rous sarcoma virus: purification of the virus and isolation of the nucleic acid...

...RESCUE in therapeutic applications, the authors tested various dRanCas13b truncations that would allow packaging...longer stretches of duplexed RNA. Using an ADAR2DD catalytic mutant, they showed that editing is mediated by...into adeno-associated viral vectors, they tested truncated ADARDDs to see if they could shrink the construct... to deaminate cytidine and used dRanCas13b (catalytically inactive Cas13) to target the cytosine deaminase...construct for viral delivery. They found C-terminal truncations allowed the same or improved editing ability ...

...Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate natural cellular...researchers can now select the best tool for their application from among many options.  Conclusion With these...engineered human uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467...via engineered uracil-DNA glycosylase. Nature Communications, 15(1), 6397. https://doi.org/10.1038/s41467...

... they are based on light which is very easily scattered by tissue. Ultrasound, on the other hand, is a...protein nanostructures called gas vesicles, which scatter sound waves and thereby produce ultrasound contrast...limiting their applicability. They also did not scatter ultrasound nonlinearly, making them difficult to...out to develop next-generation bARGs that could scatter ultrasound nonlinearly and be easily expressed ...promoter pBAD and a low-copy number origin of replication (p15A) worked best under a wide range of inducer...smaller genetic footprint, and nonlinear acoustic scattering. The best candidate for this was the Anabaena...

... Aptamer biosensors are used in a variety of applications, including detection of environmental pollutants... opens aptamers for use in many entirely new applications including aptamer based fluorescent biosensors... render this approach less sensitive in most applications. Learn About Fluorescent Biosensors Light-up..., MG proved to be too toxic for many in vivo applications, but this work proved the concept that RNA could...and biochips. Ultimately, there are just some applications that lend themselves to aptamer-based instead...Kyung-Mi, et al. “Aptamers and Their Biological Applications.” Sensors (Basel). 2012; 12(1): 612–631. PubMed...

...a protein’s stability or post-translational modifications, or study how a target protein interacts with...Protein A and Protein G are not suitable for all applications, such as protein isolation from serum. In this...capture antibody interaction and/or the downstream application. Typically, if the target protein will be detected...and an isotype control antibody. Alternative applications Oftentimes, an IP-appropriate antibody simply...identify DNA binding proteins involved in histone modification. Similarly, RNA IP (RIP) can be used to isolate...resources References:  Björck L, Kronvall G (1984) Purification and some properties of streptococcal protein...

...positively charged polycation, polybrene. Interestingly, one study found that alternative polycations such as DEAE-dextran... freshly collected lentivirus for downstream applications. While this ensures that the highest titer virus...lentivirus may want to consider testing a variety of cation and serum sources to ensure the best possible transduction...establishes the best conditions for specific applications, stick with them! Changes in such variables ...efficiency of lentiviral vectors: effect of sera and polycations.” Molecular Biotechnology 53.3 (2013). 308-14...

...EthoVision. Consult the literature to find all the applications of optogenetics relevant to your field. Ultimately...Inhibition of neuronal activation is a little more complicated since basal neuronal activity in your brain region...technology is improving and may one day reach the sophistication of mouse transgenics. Everything that has been... and memory in opioid addiction and science communication/science writing. Derek is also a pianist and...rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement." Neuron 72.5...sensitivity optical neural silencer: development and application to optogenetic control of non-human primate cortex...

...advice on how to dive into new forms of science communication. Click here to listen to the interview with ... from Addgene.      Check out more science education tools like Verse Two and Fill in the Planck at...with us. Tom McFadden is a YouTuber and science educator looking for creative ways to implement the next...next generation science standards and communicate science. Learn more about Tom on his youtube channel ...Community Check out All of Our Podcasts Find Great Educational Content in our Plasmids 101 Series Additional...