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... 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...

...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...

...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...

...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...

...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...

...may contribute to more in the future. Figure 1: A timeline of CRISPR base editing advances that led...Lander, E. S. (2016). The Heroes of CRISPR. Cell, 164(1), 18–28. https://doi.org/10.1016/j.cell.2015.12.041...Advancing from this invention to KJ’s clinical application in under a decade is an astounding feat of public...effectiveness varied depending on the chromosomal location, sequence context, and cell type. Multiple labs...of identifying a successful editor. For this application, ABE8e turned out to be most effective. Targeting...editors with increased activity and therapeutic application. Nature Biotechnology, 38(7), 892–900. https:...

...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...

...deletions, and all possible base-to-base changes.   1. Plasmids 101: Transformation, Transduction, Bacterial...resources can now be found in one place: the Educational Resources page. So let’s get to it. Here’s the...Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins by Cary Valley,...protein size and shape, linker sequences, and fusion location, there are many choices to make. 2. Prime Editing...Resources at Addgene Browse the repository Find educational resources at Addgene Search for plasmids at Addgene...

...Zhang Lab's Nature Protocols Publication describing them here. References 1.Ran, F. Ann, et al. "Genome...

...2013 edition. 10 new developments in open science 1)      In February, the Office of Science and Technology...support for open access to peer-reviewed journal publications, although many don’t necessarily publish in ...

...P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks to histones in promoter...lentiviral transduction. Lysine-specific Demethylase 1 (LSD1) Tatjana Sauka-Spengler's lab has deposited ...epigenome (the sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting...DNA sequences. Non-editing CRISPR applications direct catalytically dead dCas9 fused to a variety of epigenetic... the catalytic domain of p300 acetyltransferase increases levels of H3K27ac histone modification at specified...Preps for Epigenetic Modification Why use epigenetic modifiers? Epigenetic modification is certainly not ...Nature Communications 8: . https://doi.org/10.1038/ncomms16026 Find plasmids from this publication at Addgene...

...to western blotting  In a western, proteins are: (1) separated by size, (2) transferred to a membrane,...  (3) detected using antibodies.   Figure 1: Overview of the Western blot process. The sample,...post-translational modifications, quantify protein levels relative to standards, detect the cellular location of proteins...pull-down assays.  Westerns are divided into two categories, native and denaturing. In a native western, ...matrix, the blotting membrane, the gel, a copper cathode/iblot gel matrix, and filter paper. Pretty much...search, first pay attention to the validated applications listed on the vendor’s website and choose an...western and vice versa.  In addition to validated applications, pay close attention to the controls listed ...

...target cell line or organism (Presented in figure 1). More information on Agrobacterium-mediated transformation...mediated cloning of your gRNA of interest. Figure 1: Simplified schematic of agrobacterium-mediated transformation...current CRISPR research focuses on its potential applications for human medicine (Waltz, 2016), the potential...engineering, on the other hand, allows for targeted modification of known or suspected genes that regulate a ...model organisms like Arabidopsis and Medicago truncatula and several crop species including potato, corn...engineering using CRISPR, highlight the specific modifications to CRISPR machinery that allow for the use of...CRISPR principles). However, plant-specific modifications to commonly used CRISPR plasmids are necessary...

... and deep-brain fiber photometry.  Figure 1: Characterization of iGluSnFR4 in mice. A) Scheme ...CycT1 or PolII-CTD antibodies and unlabeled MS in DLD-1 cells (colorectal cancer). Image courtesy of Brianna...Here's what you'll find in this post: Glutamate indicators with increased sensitivity and tailored deactivation...viral preps targeting interneurons Glutamate indicators with increased sensitivity and tailored deactivation...popular choice for a variety of synaptic imaging applications. Find iGluSnFR4 plasmids here! Aggarwal, A...., Iyer, R., … Podgorski, K. (2025). Glutamate indicators with increased sensitivity and tailored deactivation...DNA vector that contains everything needed to replicate inside a host cell but with space to insert your...

...resume that will give you a chance at an interview:  1) Have an excellent resume ready to be submitted The...demonstrate in your application). Beware the myths of scientist “overqualification” but it may not be ...Utilizing existing relationships as part of the job application process can dramatically increase the chances... of a resume can be crucial for a successful application. It takes time to prepare an excellent resume... here for more on timely submission of a job application. 3) Read job descriptions carefully Be open minded...bold. You don’t have to have all of the listed qualifications to apply for a position.  A job description...target someone in a specific department and/or location. The more people you meet the better chance you...

...and drawbacks of four split AAV vector approaches. 1. Overlapping  Overview: The overlapping strategy has...Adeno-Associated Virus Capsids. Human Gene Therapy Methods 27:1–12 . https://doi.org/10.1089/hgtb.2015.140 Frischmeyer...that the oversized AAV genomes are fragmented or truncated when packaged and then following transduction,...and may need to be optimized for each unique application (Trapani et al., 2015).  Expression of smaller...transgene to help prevent the accumulation of such truncated proteins. Examples of use:  Ghosh et al., 2008...design, donor A may be capable of expressing a truncated protein. This can be avoided by excluding a stop...JS (2011) Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination Using rAAV6...

...advocacy, and writing legislation (see Figure 1 for career map). The beautiful part of science policy is...about science policy as a profession. Figure 1: Science policy can lead you to many paths! For a ... policy implementation, program management, communications, diplomacy and international relations, advocacy...they train and bring researchers to Congress to advocate for increased science funding and other issues...sets for a science policy career is science communication so an outsized way to prepare is to do lots ...Initiative.    Get practical experience in science communication by writing for the Journal of Science Policy...connect you with alumni before and during your application process so you can learn more! Figure ...

...et al., 2019; McLaughlin, 2019).    Figure 1: Schematic of multiplex immunofluorescence approaches...). Springer New York. https://doi.org/10.1007/978-1-4939-8935-5_26 Jambor, H., Antonietti, A., Alicea,...protein is (localization) and how much there is (quantification). But what if you’ve got several interesting...primary antibodies that have been validated in IF applications (find IHC validated or ICC validated antibodies...informative image-based figures for scientific publications. PLOS Biology, 19(3), e3001161. https://doi....Immunocytochemistry Protocols for other antibody applications...

...organized and boost your productivity in 2014. Tip 1: Use color coding, labels, and post-its to make finding...moving your paper spreadsheets for reagent storage locations, such as the list used to organize your cryo freezer...

...Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed PMID: 15026169. Soo, Rochelle M., et al... into a pulsating display imitating a moving caterpillar. The worm controls the snail’s behavior, forcing...exposed to predators. Hungry birds spots the “caterpillar” and then poke out the snail’s eyes and ingests...

...information Since the inception of Phage Directory ~1 year ago, Sacher and Zheng found that they could expand...researcher may only know how to prepare phage for one application but not another or they may have only used phage...Directory expands, it can also fill this role by cataloging and archiving information on individual phages...

...strategies in the comments section!   References 1. Yearly Planning Meetings: Individualized Development...determining meaningful solutions. PI-trainee communication builds a positive lab culture Although they ... communication...

...Kan, SpHIS5, or CaURA3 selection marker. Lee et al.(1) assessed many of these fluorescent tags in S. cerevisiae...insert will typically contain both a protein modification sequence and a selectable gene product for isolation...tags have been integrated. Simply design your amplification primers with the desired targeting homology—...cerevisiae, looking at their performance in categories such as brightness, stability, and disruption of...optimal FP combinations for use in yeast imaging, categorized by specific filter sets and experimental output...This collection is ideal for tandem affinity purification of protein complexes. John Pringle and Jürg ...deposited a large collection of plasmids for genome modification in yeast developed by Dr. Pringle’s former lab...

...literature? Very often the data of interest are in 1 or 2 figures from one paper and then another figure...include: Cataloging a reagent’s performance as in this antibody ReFigure Analyzing replications as in this... ReFigure. Reading and exploration including replications and experiments resulting in “negative data”...small selection of data found in full-length publications. As early career researchers, we wondered how...reagent performs well, or you might publish a replication experiment that you conducted as part of setting...papers as they are not included in traditional publications. By submitting your data to a repository (Figshare...-registration, preprints, data, software, micropublications) in grant proposals. ReFigures are one such...

...datasets by computational biologists. Figure 1: The BioGRID Open Repository of CRISPR Screens (ORCS...   Figure 3: Each publication featured in BioGRID ORCS has a dedicated summary page with PubMed...favorite gene was described in a CRISPR screen publication. You see this mentioned in the results section...manually curated and annotated from more than 45 publications, beginning with the seminal research articles...screen curated in ORCS is linked to the original publication via its PubMed identifier, which can also be ...a list of individual screens curated for the publication. Clicking on the screen name takes users to a... supplemental data tables available with the publication are also provided for the user. This approach...

... everyone was very approachable.     Fig. 1: Examples of DNA sequencing QC work    In addition...working in your chosen field. This cooperative education experience (co-op) is what has brought me, a second... QC/QA team as a DNA Sequencing Intern. The application process When I first saw the comprehensive job...for the intended position. After submitting my application, I was contacted to participate in a brief fifteen...the depositing labs intended. With the use of applications like SnapGene and Geneious, I was able to determine...currently working on projects for the Science Communication function at Addgene. Working a 9-5 As it turned...

...social media? No problem - see our recap below! Day 1: gRNA design tools: How to Design your gRNA for CRISPR...job - we strive to also give you high-quality educational resources to help you do better research. CRISPR...design, sorting the plasmids we have available by category. Each plasmid page now has a helpful graphic to... newest page in the Transcriptional Regulation category already has five blue flame plasmids covering ...providing an avenue for temporary/reversible CRISPR modification. In addition to these subpages, our updated ... HypaCas9 Day 10: Transcriptional regulation: Truncated gRNAs for Regulating Gene Expression and CRISPR...

... if the name was the correct one!)    Fig. 1: HeLa cells. Image courtesy of Josef Reischig.  ...compounds, links to publication references, and cross-references with cell line catalogs and collections....surveying of cell line collections websites and catalogues Literature searches utilizing Google Scholar ...include accession number, species, and assigned category (cancerous, hybridoma, etc.), as well as sex and...manually going through the pdfs of the 26,000 publications cited. If the information seems incomplete or...

...of experiments they are commonly used for.  Table 1. Points to Keep in Mind When Performing Biosensor ..., and you should always check the associated publication for the specifics of your chosen plasmid. Types...conformational changes, translation, and protein translocation. Targeting your biosensor Before expressing ...Experiments Experiment  Detection Method Notes Quantification of a process Plate-based assays (plate reader...components of individual cells Separation/identification of fluorescently-labeled components Fluorescence-activated...

...plasmids. The Zhang lab, on the other hand, uses a 1 plasmid, single infection system. Genome wide mouse...  “This enables you to do customized genetic modification on a scale that was really not possible before...genome engineering with CRISPRs was limited to modification of a few loci at a time,” Wang said. “It was... lentiviral libraries and their demonstrated application offer proof-of-principle that CRISPR-Cas9 can...

...target genes, but we’ll save that application for another day! References 1. Tanenbaum, Marvin E., et al....aggregation, it’s recommended for most imaging applications. What can you do with SunTag? Tanenbaum et al...al. “A protein-tagging system for signal amplification in gene expression and fluorescence imaging.” Cell...

... successful postdoc fellowship. 1. Reverse engineer success On Day 1 of your postdoctoral experience,...need? Multiple first or corresponding author publications (published, not in preparation) A history of...The other items will follow. If you get more publications, you will be more competitive for fellowships...to write as well as mentor. Yet, even the most dedicated mentor is not always available for mentees due... project. Furthermore, Group Leader position applications require 3 letters of reference- the graduate...interests, complementary perspectives, a shared communication style. You “get” each other. The other factor...makes time for you, as needed, this is a strong indicator that this person cares about your science and ...

...plasmids CRISPRi in Caulobacter crescentus Figure 1: Serial dilutions of CRISPRi in Caulobacter cells ...expression of specific Caulobacter genes using a catalytically dead Cas9 (dCas9) derived from Streptococcus...methylation-silenced genes. Read the paper in Nature Communications Find Casilio-ME plasmids If you have a new...

...that work for our experiments!)     Figure 1: The IBEX Imaging Community front page. That’s...something doesn’t work, it’s not a failure. It’s an education.   Figure 4: Zenodo page showing the IBEX... repository for reagents, protocols, panels, publications, software, and datasets. It’s also a great place...

...Last updated on Oct 1, 2020 by Aliyah Weinstein. This post was contributed by guest bloggers, Wenning...process check out our Mouse Modeling blog posts: Part 1: Genetically Engineered Mice and Part 2: Breeding ...interested in using CRISPR to develop therapeutic applications that better human health.     References Cong...

... antibody recognizes and binds to.   Figure 1: Labeled diagram of an antibody including Fc, Fab,...45. https://doi.org/10.1016/B978-012198382-6/50026-1 Wang R, Xiang S, Feng Y, Srinivas S, Zhang Y, Lin ...tool that can be used for a variety of research applications. Within the immune system, antibodies are produced...antibodies to bind proteins is useful for research applications as well because they allow scientists to target...150 kDa, a nanobody is 12-15 kDa.   Research applications for antibodies You can use antibodies for experiments...Let’s dive in and take a look at some of these applications. Western blot Western blots are used to detect... by binding to cell surface receptors. These applications are typically used for investigating signaling...

...communities  Table 1: Examples of defining your audience for science communication activities  Many people... can and do consume science communication.  Formal education is education that is provided through a structured...underlying all communications efforts.  Scicomm definitions (as used by Addgene)    Science communication, or science...time engaging with science communication efforts.  Scientific communication or scientific writing is peer...to note that it is the actual communication itself, not the communication method, that determines if something...scientific or science communication. A Tweet is still considered scientific communication if the language is... engage or disengage at will.  Informal education is education that is provided, typically ad hoc, for...

... towards understanding this disease. Figure 1: Scientists are continuing to do essential research...funding and volunteer opportunities, protocols, publications, and more to accelerate the pace of research...

...depends on what you are looking to study. Figure 1: A schematic showing the differences in chemogenetics...beneficial for use in therapeutic or behavioral applications where drawn out effects are required. Targeted...

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

...proteins, DNA, and RNA used in CRISPR.   Figure 1: Overview of the parts of CRISPR. The bacterial chromosome...simplifying the CRISPR-Cas9 system for laboratory applications. At Addgene, we sometimes refer to the programmable...

...the life scientist or bioengineer.   Figure 1: Map of a plasmid with its elements described below...Description Origin of Replication (ORI) DNA sequence which allows initiation of replication within a plasmid...point for PCR amplification or sequencing. Primers can be exploited for sequence verification of plasmids...important. Our “Plasmids 101” series designed to educate all levels of scientists and plasmid lovers - serves...plasmids are small circular pieces of DNA that replicate independently from the host's chromosomal DNA....virulence. All natural plasmids contain an origin of replication (which controls the host range and copy number...Minimally, lab-created plasmids have an origin of replication, selection marker, and cloning site. The ease...