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...Addgene started distributing a small but growing catalog of ready-made AAV and Lentiviral preps. This new...directions. I would like to thank them for their time, dedication, advice and encouragement. Why virus? Why did...performs extensive quality control, generates  educational materials, and provides viral vector technical...answering questions on virus and helping provide educational resources on this collection. With their expertise... resource and providing fantastically useful educational content on viral vectors.  How Addgene’s Viral...about the new initiative and serving all the new educational content via the Addgene Blog and our expanding...the impression that it took a lot of work and dedication to do this well ... because it did. So finally...

...to the right indicates a higher signal, or upregulation, and a peak to the left indicates a lower signal...anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the percentage...case for you, switching to a contour plot should indicate where the two populations diverge. The smear ...place your gate based on the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing...Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes it easier to identify and study...addgene.org Addgene's Antibody Guide rAb Affinity Purification Protocol Video rAbs in the Addgene repository...

...editing applications.  Figure 4: Overview of CasMINI for genome engineering applications. Image ...stability in cells, and use it in a variety of applications. This set of new anti-FLAG frankebodies includes...to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling various targets... and anti-HA frankenbodies have a variety of applications, including live-cell imaging, single-molecule...members or students new to protein expression and purification. Figure 3: The Penn State Ladder run ...challenges for in vivo delivery and cell engineering applications. CasMINI, a new miniature CRISPR-Cas system ...

... one half of naturally occurring restriction modification systems that prokaryotes use to protect themselves... has genes encoding one or a few restriction modification systems containing methyltransferases that add... phage for instance). Aside from restriction modification systems, DNA methylation also plays an integral...integral role in regulating genome replication, repairing mismatched basepairs or small indels that occur...methylases) are independent from the restriction modification systems, yet can still affect whether certain...specifically associated with any restriction modification systems, their sequences may overlap with restriction... Additional Resources Review of restriction modification systems NEB's Resource describing Dam and Dcm...

...competitions. After much learning, the focus of the application was changed, but Sample6 still leverages the ...fit for this set of technologies, effectively communicating the advantages embodied, and driving the enterprise...He explained that entrepreneurs must clearly communicate their idea at various levels of complexity and...backgrounds how amazing their idea is. While communication styles in academia and industry vary drastically...biggest challenge during this time was learning to communicate effectively to non-scientific audiences. "There...for the company; this will help avoid future complications and potential conflicts that waste money and...succeeding. Take home Starting a company requires dedication, passion, and around-the-clock work. Mike wraps...

...voltage indicator Article contributed by Shreya Vedantam Genetically encoded voltage indicators (GEVIs)...fluorescent optics. Existing red fluorescent voltage indicators, however, fall short compared to GFP-based sensors...linker between them. The Ace-mScarlet voltage indicator works like many FRET-opsin GEVIs. With an increase...channelrhodopsins. Find the Ace-mScarlet voltage indicator plasmids at Addgene! Beck et al., Sci Report 2019...increasingly been recognized for its biotechnological applications including conversion of CO2 into water and electrical...the N-terminus of Cre (CreN) are fused to the truncated Phytochrome B (PhyBΔ) and the active protein binding...

...re used in a number of research and clinical applications. Most of the time, when someone speaks about...deeper penetration into a tissue, useful for applications like IHC, or be able to access an epitope folded...that would size-exclude an antibody. In imaging applications, smaller affinity reagents means higher resolution...antibody’s ‘Y’. They are often used in clinical applications. Fab fragments can be created in lab in situations...Shahied, 2004). They are often used for clinical applications, including imaging.  Diabodies are ~60 kDa proteins...antibodies are still an excellent option for many applications, and frankly, if ain't broke, don't fix it!  ...flexible in your approach to affinity reagent applications. Whatever affinity reagents you use, one thing...

...enough space! There’s enough space here at our new location to grow. We are hiring again and getting ready...to keep up with the demand with excellence and dedication. We don’t have worry or wonder if providing...research, hosting conferences, and are expanding our catalog of materials to better serve the community. We ...projects allows us to support more new projects. Diversification is happening. The Addgene team will have some...we might do. I am grateful every day for their dedication, flexibility, innovative thinking, and openness...Addgenies support a high collaboration, high communication, high efficiency, high quality, low meeting,...about the Addgenies. I never worry about their dedication, excellent intentions, or incredible drive to...

...changes in expression of TRIM21-associated genes, indicating that TRIM21 overexpression does not aberrantly...light microscopy, comes from his recent Nature Communications article “SRpHi ratiometric pH biosensors for...250 nm), and studying endocytosis is further complicated by the reliance on pH-based biosensors used to...the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors to meet all of them. These...SRpHi Biosensors here. Richardson et al. Nat Communications. 2017. PubMed PMID: 28924139. FIRE-Cas9 ...system to study epigenetic modulation. While catalytically inactive cas9 (e.g. dCas9) has been fused to...plasmids can be found here. Braun et al. Nat Communications. 2017. PubMed PMID: 28916764. Plasmids for...

...summarized in a recent Nature Medicine paper. In the publication, Bhang and coworkers used the ClonTracer library...propagated it, separated it into roughly identical replicate groups, and treated these groups with the same...below): 1. Resistant cells among the separate replicates would contain the same labels - i.e. they would...subpopulation 2. Resistant cells among the separate replicates would have different labels - i.e. they would... Looking at barcode abundances among multiple replicate populations, they found that similar barcodes ...populations were shared by at least one other replicate; prior to treatment, there are small (previously...such that initial resistant populations can be eradicated up-front quickly. This approach is in contrast...

...regulation thats as sophisticated as a natural network. We could build a material as sophisticated as wood. To... perspective and you can layer them for more complicated functions. Those things are easy with CRISPR....submitting parts from another paper for resource allocation. Those will allow you to set the amount of transcriptional... carbons and oxygens. Nothing too much more sophisticated. There are also proteins, like antibodies. But...just using what biology has given us. Wood is a complicated material and we can access it, but we are stuck...able to design new materials and chemicals as sophisticated as those that living cells and animals make....

...in the brain region you wish to stimulate 2) Fabrication and implantation of the optical fiber into the...Channelrhodopsin2 (Chr2), a blue-light sensitive non-specific cation channel, activation of which results in depolarization...optogenetics. Successful stereotaxic surgery relies on identification of the bregma and lambda sutures and subsequent...bregma which allows you to quickly and reliably locate your injection site. We recently purchased this... fluorescent protein, which allows for easy identification of opsin expression, extent of viral spread...express for too long (synapsin was privately communicated to me as one of these extremely high expressing... and memory in opioid addiction and science communication/science writing. Derek is also a pianist and...

...visit the Cell Catalog on the Allen Cell Explorer, where you can also find the certificate of analysis provided...signal that makes it hard to visualize the precise location of the protein or structure of interest. Endogenous...technology, inserting large tags into a precise location in the genome is still inefficient, particularly...from these cell lines can be found in the Cell Catalog on our data portal. The plasmids should be functional...

...containing genes with many modifications. We look for point mutations, truncations, and insertions that could...significant amount of QC. First, we perform Picogreen quantification on each and every isolated plasmid sample and... preparation process and includes Picogreen quantification of various intermediate steps, a sizing electrophoretic...electrophoretic gel, and qPCR quantification of the final libraries. A broader view of the whole plasmid...often find a few mismatches in the origin of replication or other common backbone elements. Since we’ve...annotated Genbank files that are useful for more complicated plasmids, like those that contain synthesized...

... from E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  ...accidental insertions and deletions (indel) by the catalytically impaired D10A nickase from ABE7.10 can be a ...the authors created ABE8 constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The...these variants, ABE8 could target many additional locations in the genome. Compared to ABE7.10, NG-ABE8.xm... reduced off-target editing frequencies. For applications that require high DNA editing specificity, for...deamination, an important attribute for therapeutic application. ABE8s demonstrate an overall improved base editing...editors with increased activity and therapeutic application. Nature Biotechnology. https://doi.org/10.1038...

...channelrhodopsin, fused to a fluorescent GFP marker (CatCH-GFP). The lab deposited AAV plasmids containing ...expression. Surprisingly, <1% of synthetic promoters replicated the expression specificity of the wild-type promoters...reproducibly labeled retinal cells can be found here.  Applications for synthetic promoter AAVs Besides restricting...synthetic promoters have a few other potential applications: Using promoter combinations for targeted expression... promoters expressed in. The lab tested this application using one promoter to drive expression of Cre-mCherry...drive expression of a Cre-dependent fluorescent CatCH-GFP reporter. Expression of GFP will only result... rates of labeling are ideal of gene therapy applications, promoters that produce sparse labeling would...

...flexibility in the location of the cut and subsequent edit. In these applications, location takes priority...make a choice about what is your top priority: location or efficiency. If you are just trying to shred...still leave you choosing between efficiency and location.   Nearly PAM-less editing   The Kleinstiver lab...in double stranded cleavage and base editing applications, with high editing capacity in both contexts...randomly selected genomic loci will have a PAM located next to it. The SpRY Cas9 can also work with NYN...

...development programs for bioscience educators. Using these hands-on tools, educators introduce their students to...biologists have always struggled to effectively communicate the nuances of protein structure in a flat 2-...dangerous. Subscribe to CRISPR Blog Posts How do educators and students use molecular models?   We created...something tangible. Under the guidance of a trained educator, these instances of focused attention become magical... their labs. Soon after we began working with educators in our Center for BioMolecular Modeling, we spun...through flexibility It is common knowledge in the education community that All models are wrong….Some models...

...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration... an enhanced pU6cm promoter, viral replicon amplification of cargo, and heat treatment.  Figure...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...and more responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5 can be imaged...The initiation site identified in panel (C) is indicated; red line is exponential fit. Adapted with permission..., M. Z. (2024). A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...activation triggered by endogenous GPCR. Example applications include large-scale pharmacological profiling...

...setting—especially if job applications require teaching experience or high numbers of publications, which may take...their funding. Communication: In both industry and academia, you will have to communicate your science ...people involved is staggering. The process is complicated. If you’re interested in the drug discovery process... proprietary compounds or information. Where publication is expected, the process is similar to academia...science to others and advocate for the importance of your work. In my experience in industry, you’re more likely...

...stem cells are: Their ability to self-renew or replicate while maintaining the undifferentiated “stem” ...Unipotency: As the name suggests, cells under this category can differentiate into only one cell type. Muscle...differentiate only into myogenic precursor cells [5]. Classification of stem cells Stem cells are classified into...somatic stem cells, these multipotent stem cells are located in small quantities in specific “stem cell niches...body. These generate specialized cells for their location, mostly to maintain cell numbers or replenish ...joined Cell Press to pursue a career in science communication and publishing after completing her postdoctoral...Extraction of Pluripotent Embryonic Stem Cells: Implications for Regenerative Medicine and Reproductive Medicine...

...experts in science communication in real time! I follow great online science communicators like Phil Plait...the public, so I reached out to people who were educating the public and organizing events. I contacted ...about any topic I like and gain experience in communication, editing, marketing, and website organization...when I was trying to find local informal science educators, and ever since, the State Museum has been the.... She is particularly interested in science communication and fundamental molecular biology.      Additional...  science communication...

... unique compared to the rest of the genome and located immediately 5’ to a protospacer adjacent motif ...coli, and T. gondii genes.  2) What genetic modification are you trying to make? Addgene carries CRISPR...next-generation sequencing to check the quality of your amplification. If you've obtained a ready-to-use lentiviral... As such, sequencing CRISPR libraries after amplification and after a screen requires the use of next-...experiment, the use of appropriate controls, multiple replicates and several cell types can strengthen your results...independently validated to ensure that the desired modification produces the phenotype you screened for in the...information on CRISPR screens check out Addgene’s 2016 publication “Practical Considerations for Using Pooled Lentiviral...

...excited to announce we now offer an export license application for rabies materials! Some of the materials in...export-controlled materials in the USA, where Addgene is located.  The export license, if granted, allows us to ...international requestors. We also offer export license applications for VSV and SARS-COV-1 plasmids. Export control... applied to cover processing costs. Once the application is approved, Addgene then ships the material ...offered VSV and SARS-COV-1 plasmids under this application process since May 2020. We find this process ...whenever possible, as we cannot expedite the application process.  More information about export control...

...subfields.  Plasmid requests correlate with paper publication The dataset includes 11,426 plasmid requests ...14%). Spikes in plasmid requests correspond to publications of popular papers. Figure 1: Plasmid requests...effect on plasmid requests Before we began any sophisticated analyses, we first visualized our data in various...orders and new COVID-19 cases for each week by location. We observed that as expected, plasmid orders ...independence assumption of the Poisson process. This modification also eases the interpretation of the parameters...effects fairly well. However, we observed one complication in the China data – there is a period of 1 month...fundamental scientific discovery and high-impact applications.       Alvin Hsu is a graduate student at Harvard...

...containing a phage origin of replication that can be packaged into replication-incompetent phage particles...essential processes: cell wall synthesis, DNA replication, or protein synthesis. Because they target such... and medical organizations are working to help educate the public about proper antibiotic usage. Although...mammalian viral vectors in that the vectors are replication-deficient. Thus, a very large number of phagemids...CRISPR antimicrobials combines them with both replication-competent phages and antibiotics. Phage therapy...bacterial host range. To further potential clinical applications, Ando et al. recently engineered synthetic phages...

...transgenic mice. Purification: GFP can be used as a general epitope tag for protein purification and a number...their experimental needs, and some of the common applications in the lab. Why Green Fluorescent Protein?...to maintain fluorescent activity; very little truncation is tolerated, however, point mutations are acceptable...Re-engineering GFP to increase its range of color and application Almost as soon as its sequence was elucidated...others) Neutral mutations    A multitude of applications Due to its size and ease of use, GFP and other... just scratched the surface of the potential applications for GFP. It has also been used to identify particular...

... are not ideal for every application. Disadvantages include their duplicated (i.e., redundant) genome,...mutant, which regulates a developmental pathway implicated in congenital heart defects and cancer. These...DNA into the genome is useful for a variety of applications. For example, specific tissues such as blood...pathways control this process. Additional zebrafish applications and conclusions Zebrafish are also popular outside...—have proven useful in a variety of research applications. Lineage tracing studies highlight how these...Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments." Methods...

...efficiency. This can be done through domestication, which is the modification of a DNA sequence to remove a ...with every Addgene MoClo kit through their online catalog pages. We guarantee this will save you a lot of... the synonymous mutation of a codon. You can domesticate the sites yourself using PCR, but gene synthesis...gene synthesis, you can request the sites to be domesticated through the process, and request your sequence... overhangs for future assemblies in the right locations.   We hope these tips will help you in your MoClo...Modular Cloning Plasmids 101: Modular Cloning Applications and Kits Plasmids 101: Golden Gate Cloning ...

...Addgene has a growing catalogue of viral preps available to request. In this post, we’ll walk you through...minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties...posted on the material page for the corresponding catalog item (see maps section for images).     ...Lentivirus Production Lentiviral preparations ("-LV” catalog items) are produced in 293T cells using the 2nd.... Concentrated lentiviral preparations (“-LVC” catalog items) are generated from lentiviral preparations...typically prepared using endotoxin-free plasmid purification kits.     Figure 4: Schematic of the... Addgene's Data Hub Digital Droplet for AAV Quantification Viral Vectors 101: An Introduction to AAV References...

...control, 1; very dark). Brighter fluorescence indicates a more successful gRNA design. Adapted from Mashiko...plasmid using standard cloning techniques (The XX indicates where your target is inserted, disrupting the ...effective ones to increase your chances of gene modification in vivo. Find Plasmids for Testing Your gRNAs...et al., 2014). The plasmid is sturdier than the delicate RNA and therefore there has less chance of contamination... the validation assay should give an accurate indication of how many pups will contain a mutation and ... months from the day you decide to do a gene modification experiment in mice to the day you have your ...reproductive biology and genetics. She loves to communicate the latest and greatest in scientific content...

...compartments undergo significant post-translational modifications including glycosylation, disulfide bond formation...are equally susceptible to these non-native modifications, which can all potentially affect function [...N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering the protein... non-optimized parental proteins. Our recent publication illustrated the quantitative increase in fluorescent...and Jack Griffith, her research focuses on the replication machinery of Kaposi’s sarcoma-associated herpesvirus... for diverse cellular environments." Nature communications 6 (2015). Pubmed PMID: 26158227. Pubmed Central...pathway."Biochemical and biophysical research communications 430.3 (2013): 1114-1119. Pubmed PMID: 23257162...

...procedure enables quantification of antibody performance in major research applications. eLife, 12, RP91645...that the antibody being published doesn’t always indicate the presence of supportive data. To be supportive...that the antigen being detected in the intended application — here, flow cytometry — is the desired target...fixation-permeabilization protocol. Although the expected location of the target is an important factor in deciding... saponin. 400,000 cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb...this approach should also allow for the clear identification of the cell types of interest. By correlating...of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. eLife...

...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,...

... citations for recent articles. Of course, a publication in Science or Nature is always nice, but in the... lesser-known journal as it is in a top-tier publication. Accessibility is surely also behind the increase...increase in citations for older publications. (As an interesting aside, Nature recently reported that the...in a solution.) But are citations and journal publications really the best way to measure impact? As that...and what it’s made of. Increasingly, journal publications alone aren’t enough. Scientists are sharing ...consider and quantify the impact of scientific publications. Ventures like Impact Story seek to find ways...

... 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...

...efficiency constructs are great for in vitro biocatalytic applications where toxicity doesn’t matter. Plus, for... binding of the plasma membrane. In previous publications, they described tools to control formation of.... Restricting the effect to a specific time or location can be critical when, say, the gene is essential...that trap the fusion protein away from its usual location, preventing it from carrying out its proper function...) and to introduce unnatural head groups for applications like fluorescent labeling via click chemistry...substantial PA accumulates in the plasma membranes, indicated by the PA probe colocalizing with SuperPLDx30....

... do need multiple first/corresponding author publications in quality journals. It helps to come from the...a job. The currency of scientific success is publications and grants. 2. Write succinct and easy to understand...humbling. My conviction that I had a flair for communication was rapidly dispelled. She would ask me, "Is...succinct. Slide decks leads to the opposite and can suffocate an exciting idea in a swamp of unnecessary details...essential for human health. Failure to do so is implicated in several human diseases. Proper patterning ...disease treatment, etc., again in clear terms. "Implications for human disease" is vague. Instead, consider...interviews. For point of reference, I submitted my applications all by snail mail and only had phone interviews...

...decades. Widely used across various research applications, they have become indispensable tools for manipulating... easily, without the need for time-consuming purification processes, and are powerful, due to their ability...-lasting changes in cells. However, for some applications, integration might be considered a double-edged...dividing cells because they lack an origin of replication (Banasik and McCray, 2010). However, IDLV genomes... certain types of clinical or basic research applications. Considerations for IDLV use If you’re reading...Integrase-defective lentiviral vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org...lentiviral vector: prospects for safe clinical applications. PeerJ, 10, e13704. https://doi.org/10.7717/...

...aptamer soup by exploring the development and applications of fluorescent RNA aptamers in Addgene’s collection...target cellular RNA to specific intracellular locations using Cas9 and track the localization of the complex...biotin (the addition of biotin allows for the purification of specific RNAs from cells using this tag, ...TO1-biotin and TO3-biotin contain an acetate modification that reduces nonspecific binding with RNA by...non-coding RNAs in mammalian cells." Nature communications 9.1 (2018): 656. PubMed PMID: 29440634. PubMed...fluorescent rna aptamer spinach2 for imaging of replication and immune-mediated changes in intracellular ... Shanker Shyam S., et al. "Ribonucleoprotein purification and characterization using RNA Mango." RNA23.10...