... edit.   Figure 1: Choosing the best PE6 variant for your application. Image reused form Doman...delivery by modular envelope design. Nature Communications, 14(1), 5141. https://doi.org/10.1038/s41467-023... CreVLPs, and eVLPs for in vivo and in vitro applications.   Figure 2: Modular features of DIRECTED...approach for experimental therapies and medical applications.  Find DIRECTED plasmids here! Strebinger, D...interactions or map a local proteome, but some applications have been challenging or inaccessible. The Ting... (which had therefore been trafficked between locations). Figure 3: A) LOV-Turbo becomes active...active when illuminated with blue light, catalyzing the biotinylation of nearby proteins. B) TransitID uses...

...membrane targetin sequence derived either from p63(1-29) and Lck. Image from Chertkova et al., 2020. ...Reports. 2020. https://doi.org/10.1038/s41598-020-57500-1 The CRISPR corner By Jennifer Tsang Here are a few...Fluorescent tools for mammalian cells Nanobody purification CRISPRi and CRISPRa system for hPSCs The CRISPR...CRISPR corner New from the viral service Faster catalysis after yeast-based protein evolution By Joanne ...concept, Sanchez and Ting sought to improve the catalytic efficiency of tobacco etch virus protease (TEV...activity with a fluorescent protein--no affinity purification required! The team used other molecular techniques...evolution platform to generate TEV protease variants. Catalysis by TEV protease allows a transcription factor ...

...Aptamers and Their Biological Applications.” Sensors (Basel). 2012; 12(1): 612–631. PubMed PMID: 22368488...be selected for these small ligands. References 1. Ma, Hanhui, et al. “Multicolor CRISPR labeling of... 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...

...about health equity, science communication, and public health.     References 1. O'Neill, J. "Tackling drug-resistant... to the development of resistance include (Figure 1): Over-prescription of antibiotics (for example, ...Microbes (bacteria included) are very skilled at the delicate art of survival. Existing virtually ubiquitously...of intentions. The take-home? Always take your medication as prescribed. How do we know which bacteria ...

...regulated in the medicinal plant, the Catharanthus roseus (Figure 1). The ultimate goal of our research ...activities in seed plants." Plant cell reports 9.1 (1990): 1-5. PubMed PMID: 24226366. Lee, Min Woo, and Yinong...and a vacuum is applied with a vacuum bell or desiccator. Successful infiltration can often be seen by...expression method for analyzing gene function in Catharanthus roseus seedlings." Frontiers in plant science...

...Spaghetti Monster probes at Addgene Uses of smFPs 1. Multi-channel connectomic tracers Fluorescent proteins... expanded use of smFPs in the future. References 1. Viswanathan, Sarada, et al. "High-performance probes...controlled by collections of neurons that are intricately wired into circuits through synaptic connections...photostable than GFP. In addition, antibody-based amplification of their signals is weak and increases background...more HA tags. 4. High-resolutions microscopy As indicated above, smFPs can be used with conventional microscopes...

... cerevisiae (Tuller et. al., 2010). Figure 1: An overview of peptide synthesis. A ribosome interacts...McClain WH (2000) The G·U wobble base pair. EMBO Rep 1:18–23 . https://doi.org/10.1093/embo-reports/kvd001...folding and stability, change post-translational modification sites and even affect protein function (Mauro... AP, Bilen B, Zavolan M (2013) Analysis of CDS-located miRNA target sites suggests that they can effectively...synonymous codon usage bias, and its potential applications. Nucl Acids Res 15:1281–1295 . https://doi.org...

... present in the sample (APC in the case of Figure 1). In Figure 1A, the population needs to be shifted... decrease the compensation value.   Figure 1: Compensation example with single color control. Flow...diagonal population, this is a sign of spillover and indicates that it is necessary to apply a compensation value...

...glorious trophy. Sure it’s plastic and you have to share 1 trophy among 6 people, but you’ve earned bragging ...plasmids! Each chose their favorite plasmid and replicated it. Can you guess each plasmid? Our lab paid...creative hijinks last year. They showed us their dedication to great customer support by dressing up as our...

... Table 1: New ready-to-use viral vectors in the Addgene repository  Name Serotype Category Depositor...skipping the preparation steps of production, purification, and titering. Our newest viral vector service...

...a ‘plasmid’. “p” is for plasmid. pXXXXX-XXXX Step 1: Backbone name Include the empty backbone name in ... eukaryotic cells). pBACKBONE-XXXXX Tip: For a catalog of published and commercially available empty backbones...considerations  If your insert contains a mutation or modification, this should be included in the plasmid name...

...package from Addgene.  Here are the details:   Contest 1: Deck the Lab - Classic Edition This edition of the...create a stack of wrapped gifts using life science catalog pages. Make your WFH space feel a little more festive...

...Time for the #BioSci3319 #StreakPlateChallenge part 1. Lab B needs a redo :-( @SandTBioSci #CompetitiveStreaking...blog posts on fluorescent proteins Check out educational resources on the blog Resources on Addgene.org...

... Table 1: New ready-to-use viral vectors in the Addgene repository  Name Serotype Category Depositor...

... necessary for advancing the field. References: 1. Internal initiation of translation of eukaryotic ... of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. Kim et al...desktop. The Plasmids 101 eBook is designed to educate all levels of scientists and plasmid lovers and...

...alternative detection methods.      Figure 1: The four types of ELISA are direct, indirect, sandwich...conjugated antibody is not readily available. Table 1: Overview of the advantages and disadvantages of each...staining. Direct ELISAs also lack the signal amplification afforded by secondary antibodies and thus are...

...true reproducibility. The following are critical: (1) connectivity to other data sources used in research...engagement with the Wisconsin research community were to: (1) prevent vendor lock in by making it easy to get data... both accept deposit of data associated with publications into one of five general purpose open source...

...light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2). In the dark, this peptide is caged by the...iLID nano and 800nM to 47µM for iLID micro; Figure 1). With this great affinity range, these LIDs have ...Figure 2). They are all available now at Addgene. 1 Lungu et al., Chem Biol. 2012 Apr 20;19(4):507-17....Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; 112(1):112-7. doi: 10.1073/pnas. CRISPR-Cas9 optogenetic... al., BMC Molecular Biology. 2013 August 20th; 14(1). TRICK: A method for visualizing the first round...for a variety of experimental purposes including (1) single or multiple, constitutive or inducible, gene...the years. The authors optimized these systems by: 1) decreasing the number of plasmids required for each...

...sequence on either side of the desired mutation (usually 1-3 mismatched bases) is sufficient for your primers...researcher at Cornell University.       References 1. Breslauer, Kenneth J., et al. "Predicting DNA duplex...desired modification. Finally, the positive clones are sequenced to confirm the desired modification and ...polymerase activity (for amplification), 3’->5’ exonuclease activity (increases amplification fidelity), and no... genome modifications. Occasionally, multimerization of the PCR primers can cause duplication of the primer...the desired modification have been identified (and confirmed to be free of primer duplications), final validation...and the absense of additional modifications.   Site-directed mutagenesis of Plasmids. A) Generation...

...Figure 1). Clustered events are called a population.                             Figure 1: While...done using side scatter (SSC) and forward scatter (FSC), which measure light that “scatters” past the cell... the plot that divides it into four, using the location of the cell populations as a guide  (Figure 3....be used for this purpose.  Pro tip! The forward scatter is a proxy for diameter and volume of the cell,...the exclusion of doublets and debris. The side scatter roughly measures granularity, a proxy for nucleic... will have numbers by gated populations; this indicates the percentage of total events contained in the... gated population. Heat maps are often used to indicate the relative density of cells. You can see both...

...folding, and deal with sub-optimal codon usage (Table 1) A few mutations are common to all or most expression...curated list of Bacterial Expression Systems Table 1: E. coli expression strains  Note: All strains are...expression vector cannot have p15A origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysS...expression vector cannot have p15A origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysE...Expression vector cannot have p15A origin of replication F- ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE2 (...Expression vector cannot have p15A origin of replication fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS/ pLemo...Expression vector cannot have p15A origin of replication F-, Φ80ΔlacM15, thi, lac-, mtl-, recA+, KmR Expression...

...$47,476 and the date for implementation was set for Dec 1 2016. In order to be exempted from the need to pay... these institutions (populations potentially from 1 to 5761, according to the NSF data) and therefore ...Labor’s general guide to the effect on higher education, you can see that those in teaching roles (such...frankly quite surprising. Consortia of higher education institutions submitted letters to the Department...centrally administered, which leads to many complications, including the inability of some institutions...paid. Institutional salary policies are one indicatator of how salaries may changes due to the FLSA. ...of the postdoctoral workforce. Based on our communications with and research on these institutions, we...

...older epitope tags - only time will tell! References 1. Georgieva, Maya V., et al. "Inntags: small self-structured...immunoprecipitation applications, but none of the traditional tags were suitable for both applications. To prove...from non-vertebrate systems. Preliminary testing indicated that inntags IT5, IT6, and IT10 expressed well...and the chaperone Ydj1, both of which display sophisticated interactions with multiple other proteins. Again...detrimental effects on cell size and growth rate, indicating that they compromised protein-protein interactions...These tags are easy to use in multiple common applications. Due to their limited effects on native protein...particularly in single-molecule or high-throughput applications. Perhaps IT5 and IT6 will even replace our older...

...an antibody performed in an experiment.   Figure 1: The front page of the Addgene Data Hub.   Antibody...very little data on which might work in your application. It can be quite difficult to decide on which...be searched and sorted by gene/target, name, application, pass/fail rating, sample species, antibody species... how the antibody performed in the submitted application, along with a brief description of the experiment...experiment. If that data was included in a publication, you'll find that information in the report as well... section will vary slightly depending on the application — flow cytometry, western blot, immunohistochemistry...data showing how the antibody worked in your application. The submission process is easy, with simple,...

...straight to your materials of interest!   Figure 1: The new search bar.   Pro tip! You can use a manuscript...and easily navigate to educational resources, tools, collections in our catalog, and the Addgene Help ...search bar. And if you’re in the mood to browse our catalog, you can use the links under the search box to ...scrolling up and down the page.  Figure 3: The Education & Tools menu Further down What happens if you ... mission, links to our deposit, request, and educational resources page, and previews for our three most...

...know that most cephalopods die after they lay their 1 clutch of eggs? Propagation is thus quite difficult...organism blog posts: Five Popular Model Organisms, Part 1 Five Popular Model Organisms, Part 2 ... their organismal systems for scientists and educational programs for K-12 students. You can see why I...parenting strategies and make important ecological indicators since species live in water and on land. Current...which have been used to treat heart disease. Caterpillars that eat milkweed generate cardenolides that... poisonous) for bird predators - birds eat a caterpillar once, vomit, and never do it again. Cichlids ... expensive. They are using a cool fluorescent indicator system to rapidly identify recombinants. Arthropods...

...by insertion and deletion frequency. Fig. 1: Overview of TIDE. The input is sequencing trace files...keep your product to knock-in size ratio below 10:1. Small knock-ins Is your knock-in edit small, with...how many copies you are looking for. Secondly, categorize your edit – is it any frameshift mutation? A ...enzyme screening for resistivity to digestion, indicating targeted edits.   Interested in this method...a manageable number of the higher probability locations. If you’re worried about off-target effects, you...Tao Chen, Jos Jonkers, Bas van Steensel. Easy quantification of template-directed CRISPR-Cas9 editing. (...and variant-aware in silico off-target site identification for CRISPR genome editing. (2019) Bioinformatics...

...single neurons in the living brain   Figure 1: Plasmid mix to label neuronal morphology (eYFP) and...genOway press release for more information. References 1. Atasoy, D., Y. Aponte, H. H. Su, and S. M. Sternson... gives you a single level of control without modification - either Cre is there or it’s not. Cre-mediated... sites can lead to excision, inversion, and translocation of DNA in a similar fashion as between loxP ...37 °C). Smart molecular evolution led to the identification of FLPe which has a temperature optimum of ...-LDB domains of the fusions allowing Cre/FLP translocate to the nucleus where they exert their recombinase...

...school to a startup and back to academia. Chapter 1: How working at a startup prepared Tom for an academic...foundational research, training, startups, and real applications of SynBio. Chapter 3: Synthetic biology as the...synthetic genome project and constructing ever more complicated biological devices. Chapter 6: - Making research...Addgene Blog Learn about the Synthetic Biology Education Nonprofit BioBuilder Plasmids 101: Golden Gate...

...  Table 1: New ready-to-use viral vectors in the Addgene repository  Name Serotype Category Depositor...

...activity to a transcriptional activation domain (Fig 1). Two ways to use CRISPRa for your research CRISPRa... Stucka R, Hauber J, Lange UC (2016) Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional... (2014) A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging.....org Find Cas9 Activators Find Cas9 Repressors Catch Up on Your CRISPR Background with Our CRISPR Guide...

...breaks (DSBs) or donor DNA templates.  Figure 1: An overview of prime editing. Created with BioRender.com...demonstrated ~20-50% efficiency in HEK293T cells with 1-10% indels. Since then, further innovations have continued...Engineering. https://doi.org/10.1038/s41551-024-01227-1 Yan, J., Oyler-Castrillo, P., Ravisankar, P., Ward... the indicated tool. ~ reflects an innovation that may optionally be combined with the indicated tool....genetic variants have been identified in humans and cataloged in the ClinVar database. Previously developed ...prime editing Less constrained by PAM sequence location The prime editor extends the reach of CRISPR genome...editing typically needs to be optimized for each application, and the efficacy varies widely — it still almost...

...is here to help. Contact us at [email protected] or +1 617-225-9000, or check out our MTA reference pages...results, modifications, and inventions created by the Recipient Right to file patent applications on modifications... about intellectual property rights, pending publications, and commercial use often come up. An MTA can...trials Acknowledgement in recipient’s future publications relating to the material Limited liability...

...Cloning grade DNA Transfection grade DNA Figure 1: Transformation introduces transformation grade plasmids...that have been used during the purification procedure. Good indicators of DNA purity are the absorbance...nm should fall ~1.8 - 2 and give scientists an indication of purity versus protein contaminants. The 260...nm ratios should fall ~2 - 2.2, and provide an indication of purity versus chaotropic agents as guanidine...

... successful after their training.   Figure 1: Professional Development Hub Workshop Attendees and... It's been an honor to work with such a group dedicated to providing the structure and know-how to integrate...example, pd|hub assembled a coalition of higher education organizations committed to advancing career and... inclusive career readiness training in this educational path before we lose more scientists who might...

...’ve come to the right place. Get started with PGC-1 plasmids from Addgene. References: Skeletal muscle...window onto communication between the muscle and brain, which may have therapeutic implications, say Hyo ...

... with DNA sequencing troubleshooting and analysis 1. You can use any of the following programs to view...of interest. 5. Use a silica spin column for purification of the samples you send for DNA sequencing If...requires you to perform your own Big Dye PCR amplification reaction (as opposed to using the all inclusive...

...Histochemistry & Cytochemistry, vol. 59, no. 1, SAGE Publications, Jan. 2011, pp. 6–12. Crossref, doi:10.1369...compare the experimental group.     Figure 1: A, Most biological experiments will benefit from ...results.  Replicates Replicates are another critical control in biological experiments. Replicates can be...of biological replicates such as multiple mice in each group and C, technical replicates or repeated measurements...secondary antibodies.   Application-specific controls Some antibody-based applications have very specific ...then moving on to controls for antibody-related applications. After reading, you will have a better understanding...blot and look for the molecular weight shifts indicative of tau phosphorylation. By including the positive...

...improved sensors for these and other targets. Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted...fluorescent biosensors. Nat Commun. 2022 May 25;13(1):2919. doi: 10.1038/s41467-022-30685-x. Feel the ...sensors One of the latest additions to Addgene’s catalog is a new class of sensors for neuropeptides and...doi: 10.1038/s41592-022-01597-x. New glutamate indicator iGluSnFR3 Improving on the glutamate sensor iGluSnFR...They generated variants optimized for different applications (v82 has higher affinity for glutamate but slower...PDGFR.codonopt    Aggarwal A, et al. Glutamate indicators with improved activation kinetics and localization...have developed a new genetically-encoded voltage indicator (GEVI) called JEDI-2P (Liu et al. 2022) optimized...

...detects the light scattered by that cell at a low angle (FSC) or large angle (SSC) (Figure 1). FSC values ...colleagues.   Figure 1: The laser pulse illuminates the cell and is scattered forward at a low angle... you with two measurements: forward scatter (FSC) and side scatter (SSC). To acquire these measurements...exclude all duplicate events (two cells stuck together) from your single cells, as duplicates can’t be reliably...types of scattering that occur when a cell is pulsed by the laser. Within each type of scattering, you also...to keep it short though, promise! FSC and SSC: scattering To digitally extract your cells based on their...passes through the laser beam and causes it to scatter (FSC/SSC), the detected photons can be represented...

...science’s place in education, government, and civilization in general (1). Science and politics? Science...consequences of a march. Three of these risks include: 1) Lost trust Perhaps the greatest risk is that the ...sister locations on April 22, 2017, scientists and non-scientists alike will march to advocate for science...already come to pass. It may only be by advocating for science education and literacy that this changes.  2...marching for. I will be marching to advocate for science education and literacy as I believe people that...communities, microbial interactions, and science education.  Disclaimer: The views presented in this article...process begins with a testable hypothesis - an educated guess about how some part of the world functions...

...together along with the Gibson assembly master mix for 1 hour, and then transform into competent cells. The...you should consider giving it a try! References: 1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison...neighboring fragments can be created via PCR amplification with primers that contain the appropriate homologous... tools that help you design primers for PCR amplification of fragments to incorporate such regions of ...assembly meets CRISPR Gibson can be adapted to more complicated cloning schemes, such as those where the vector...CRISPR technology and is described in a recent publication by the Lockey lab (Wang, et al. 2015). In this...aspects of the SLIC methods. First, it uses a dedicated 5’ exonuclease instead of using the exonuclease...

...example, if there are 10 putative off-target locations you can 1) determine if editing at those sites would...downstream experiments. You only successfully generate 1 null clone and proceed with it. In this case, the ...“Off-target effects” a vague catch-all term for unintended consequences in an experiment. For CRISPR ...sequencing. While this may be unnecessary for many applications, it is the only comprehensive method available...binding by Cas does not always correlate with catalytic activity at those sites, especially when DNA mismatches...For a candidate site-specific approach, mRNA quantification or western blot of putative off-targets would...you’ve tried everything listed here. For some applications, this is ok, but it still must be controlled...

...out these SciComm blog posts. References 1. Bourdeau, Raymond W., et al. "Acoustic reporter genes for... science communication and outreach - if you'd like to learn more about Science communication check out...microscopes and give a similar optical quality (magnification of 140X and 2 micron resolution). I have worked...creative approaches to visualize bacteria in an educational setting. The Symposium on ‘Creative Approaches...from hand-drawn comics (Dr. Sanja Saftic) to educational gifs created by the Amoeba Sisters. Here’s one...Bacterial Genome Engineering Learn about Science Communication using Twitter  Resources on Addgene.org Check...

... remains fluorescent in acidic organelles (Figure 1), showing that its acid tolerance is maintained in...follow him on twitter: @joachimgoedhart.   References 1. Zacharias, David A., et al. "Partitioning of lipid-modified... proteins.Therefore, GFP is not suitable for applications that require anaerobic conditions. In experiments...an additional protein module that can report on location. This approach enables the study of proteins in...fusion with GFP allows for detection of genomic locations (Chen et al., 2013). GFP fluorescence overlaps...case when proteins undergo post-translational modification with fatty acyl chains. For instance, myristoylation...C-terminal consensus sequence (-CaaX). Both modifications are disrupted when GFP is fused to these sequences...

...five methods for extracting DNA without a kit too. 1. Early Career Researcher Toolbox: Free Online Molecular...virtually. 6. Quantifying DNA? Here are Five DNA Quantification Methods to Consider by Jennifer Tsang The...The nanodrop vs. spec debate dominates DNA quantification discussions, but what about the other methods... with plasmids deposited at Addgene! 2. DNA Purification Without a Kit by Beth Kenkel To complement ...

... two years. References 1. Martz, Colin A., et al. "Systematic identification of signaling pathways with... of 17 signaling pathways that are frequently implicated in cancer cell proliferation, survival, differentiation... make the library usable for pooled screens. Applications of the cancer pathways ORFs kit Wood and his...

...infections, cancer, and other diseases. References 1. Harrington, Lucas B., et al. "Programmed DNA destruction...of dsDNA and RNA in trans. Fluorescent signal indicates the presence of this viral sequence in the assay...

...regulation of endogenous RNA. J Biol Eng. 2014 Mar 1;8(1):7. doi: 10.1186/1754-1611-8-7. PubMed.  Related Resources...says the new tool might even find therapeutic application in addition to their use in basic biology, for...

...to the interview with Science in the News Chapter 1: Introduction - Vini and Amy tell us a little about... to be crazy complicated! Listen to the full podcast for more great science communication tips or listen...tons of ways you can get involved in science communication. In this second half of our conversation with...many things you can do start your own science communication student group and get more involved with your...is a graduate student group with the goal of communicating science to the general public and training the... the next generation of science communicators. In this chapter we learn about some of their events - science... peer-to-peer feedback in effective science communication. Chapter 7: The Goal of the SITN Podcast - Vini...