...methyltransferase DNMT3A allows researchers to easily add biochemical marks to genetic regions from which where they...approach (spoiler alert: it’s dCas9… with no modifications). Ready to get started?   Figure 1:...eventually, develop treatments, we need tools to specifically edit methylation sites.  Step aside, transcriptionally... DNA. When dCas9 is bound to a target CpG, it physically blocks DNMT1 from that site. So, when DNMT1 methylates... methylates the genome during DNA replication, it doesn’t methylate sites blocked by dCas9. Within a few...Szyf 2021.   Let me leave you with a few practical considerations: Design your gRNA to overlap the...for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable, otherwise...

...process involves a significant amount of QC. First, we perform Picogreen quantification on each and every... 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...sequence or plasmids containing genes with many modifications. We look for point mutations, truncations, and...

...progress on establishing a system for the chemical replication of RNA in the absence of evolved enzymatic...enzymatic machinery (i.e. RNA replication with no proteins). The chemistry is complex and this work requires...inheritance that might be less error prone than DNA replication. He too mentioned how “surprising” small RNAs... about using CRISPR/cas to address the growing clinical problem of antibiotic resistant bacteria. See ... and Sun Yat-sen University presented two new technical developments. Using the Tet-on system, his lab...Steitz quipped that RNA in the ribosome is in a complicated conformation “not like DNA which has a boring...

...are time-consuming, have been typically optimised for immortal/genetically-corrupt cell lines, lack scalability...alone in the cellular milieu. It is, therefore, critical to understand how proteins interact with one another... another (or with DNA) to perform diverse biochemical tasks in the cell. One of the most popular approaches... to tag the protein of interest with a small biochemical “epitope tag” such that these tagged proteins... purchased from various other suppliers with a typical turnaround time of 5-7 days. The crRNA and tracrRNA... to tag >30 genes/plate in the first shot with typical knock-in efficiencies of 10-20%. Together, our ... the non-HDR allele. For N-terminal tagging, specifically searching for error-free knock-in at both alleles...

...McDonald. "A practical guide to evaluating colocalization in biological microscopy." American Journal of... proteins of interest and examine them using biochemical methods thus removing the context of the cell...way it is important to choose the appropriate quantification methods and tools. It is usually not enough...microscopy is limited by the wavelength of light and, practically speaking, for common lab microscopes the detection...Quantitative analysis of dynamic association in live biological fluorescent samples." PloS one 9.4 (2014): e94245...

... (green or blue) within a population of timer indicates the age of the pool: the higher the ratio, the...fluorescence falls within the linear window for the theoretical timer shown in Figure 1B, you can use the equation...-FT-N1 pTRE-Slow-FT pBAD/HisB-Slow-FT -  Applications of fluorescent protein timers FP timers have...protein, or cellular structure and a particular biological event (trafficking to a subcellular location,...of neural fiber bundle formation." Journal of Biological Chemistry 276.32 (2001): 29621-29624. PubMed ...to red report on cellular trafficking." Nature chemical biology 5.2 (2009): 118-126. PubMed PMID: 19136976... insulin-secreting beta-like cells." Nature communications 7 (2016). PubMed PMID: 27063289. PubMed Central...

...Because of their small size, nanobodies can be synthetically expressed off of plasmids in bacteria, and they...with a large and diverse pool of sybodies that hypothetically contain ones that bind strongly to your target...Starting with libraries and a biotinylated (for purification purposes) protein target of interest, the protocol... identifying sybodies that bind strongly and specifically to the protein of interest. The Seeger lab used...-30) of sybody candidates are sequenced for identification. All unique sybodies can be subcloned into ...2020) Generation of synthetic nanobodies against delicate proteins. Nature Protocols 15:1707–1741. https...Light to Manipulate Cell Signaling and Protein Purification RANbodies: Reporter Nanobody Fusions Plasmids...

...requires incubation at 55 °C - a dangerous, non-physiological temperature for mammalian cells and one which...been excitedly testing NgAgo's genome editing applications. So, how does NgAgo stack up thus far? A few...survey. When asked if they could detect indels (an indication of gene disruption ability) with NgAgo, one respondent...could not achieve indels with the NgAgo system indicates that it is very tricky to use and fussy to optimize...proposed in the original article, to achieve greater efficacy, researchers will have to carry out repeated transfections...results out there include: Google Groups - I've specifically set up a forum where NgAgo users can share protocols... S. Lander, and Feng Zhang. "Development and applications of CRISPR-Cas9 for genome engineering." Cell...

... most commonly used are GECIs - genetically encoded calcium indicators (e.g. GCaMP). Others include: pHluorin...capacity. Many of these FPs are fluorescent when ectopically expressed in most organisms. Furthermore, fusing...fluorescence. Therefore, FPs are used to study many biological questions. The two most common uses are: 1) to...For LSS proteins, the Stokes shift is ≥ 100nm. Specifically, these proteins are excited by UV light or blue...were prone to oligomerize. This may affect the biological function of the FP-fusion protein. Therefore,.... al. (2010) “Fluorescent proteins and their applications in imaging living cells and tissues”  Physiol...

...excited to announce we now offer an export license application for rabies materials! Some of the materials in...international requestors. We also offer export license applications for VSV and SARS-COV-1 plasmids. Export control...will need to provide a brief description of the applicable research project when purchasing export-controlled... 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...

... antibiotic resistance genes so that you can specifically isolate bacteria containing your constructs ...common antibiotic resistance genes and which applications are best (or worst) for each antibiotic. The...Ampicillin Ampicillin, often just called amp, is the historical king of the antibiotic resistance realm. Amp ...immediately after it has been heat-shocked or electrically zapped. As a result, a shorter recovery period...is expensive (relative to amp), and for some applications, the benefit it offers is marginal. Fortunately...antibiotic resistance ultimately comes down to your applications and needs. The more niche and specific your ... antimicrobial agents in MIC trays. Journal of Clinical Microbiology, 23(5), 959–961. https://doi.org/...

...translational applications (mechanistic studies and gene therapy treatments for neurological diseases). ...neuroscience research is the lack of a method for genetically manipulating neurons throughout the whole brain... creates a template that’s suitable for PCR amplification using pre-designed primers. By using this library...astrocytes. Why screen for IV deliverable AAVs? Typically AAVs are directly injected into the brain, but...throughout the body and is often difficult to surgically access. Therefore an IV injection is an ideal...pictures (see figure 4). It also enables single-cell anatomical study and neuronal tracing, aka determining where...

...that, they also help us keep track of the tubes’ physical location in the lab. These barcodes additionally...  freezers depending on the sample type. It is critical for us to know which plate, rack, and freezer ...missing so that we can make a new tube from a replicate sample. The transformation After the DNA has ...plasmid can grow. As the bacteria divide, they replicate and retain the plasmid. This allows us to make...help us track each sample, but we also have a dedicated and detail-oriented lab team that works carefully...performs an average of 140 transformations. The lab typically transforms newly deposited samples from Monday...Excel sheet.3. Be specific  - When assigning a physical location to your samples, include as much detail...

...Summer Science Communication Series. We’ll be covering the basics of science communication, tips and tricks...science with others, and introducing science communication as a research field. And there will even be ...be a few tips for scientific communication (manuscripts, talks, etc…!)  Deep Dives You probably are familiar... Dives” series comes in! We’ll take a common application or concept and go beyond the basics - diving ...you’re ready for the next steps on some common applications, join us as we get into the weeds on some of...

... gives you a single level of control without modification - either Cre is there or it’s not. Cre-mediated...37 °C). Smart molecular evolution led to the identification of FLPe which has a temperature optimum of ...asymmetric target site core but its 8 bp length that is critical for Cre and FLP function. Because recombination...Recombination between the two target sites with identical sequence leads to inversion of the flanked DNA...this plasmid mix results in sparse labeling of cortical neurons and their synapses.   How can you...control   Figure 2: Expression of a morphological marker (eYFP) and synaptic marker (PSD95-mcherry...(7): 657–62. PubMed PMID: 9661200.  3. Cardin, Jessica A. et al. 2009. “Driving Fast-Spiking Cells Induces...

...parenting strategies and make important ecological indicators since species live in water and on land....protocol community! Asclepias syriaca and Asclepias curassavica (Gerog Jander) Milkweed plants are known to many... as the only Monarch butterfly food. They have chemical pathways generating interesting drug-like compounds...Lab (@EvoEmbryo) September 24, 2018   Xenopus tropicalis (Gary Gorbsky) This is not a new experimental...plants and are unusual enough to be interesting biologically. They have economic importance as harmful weeds... expensive. They are using a cool fluorescent indicator system to rapidly identify recombinants. Arthropods...

... this post I’d like to delve deeper and look specifically at the characteristics and capabilities an ELN...facilitate true reproducibility. The following are critical: (1) connectivity to other data sources used in...likely stored in institutional and lab servers specifically designed to hold large amounts of data. Although...group’s research – it makes no sense and is not practical to move all this data in bulk into a lightweight...lightweight tool like an ELN. It is nevertheless critically important to be able to conveniently reference specific...with a particular experiment or project thus significantly enhances the ability of others to subsequently... both accept deposit of data associated with publications into one of five general purpose open source...

...expression limiting regulatory elements or use a non-canonical code (Gustafsson et al., 2004), such as Hoogsten...folding and stability, change post-translational modification sites and even affect protein function (Mauro...recognize multiple synonymous codons) may actually be critical for determining the rate of translation. The ribosome...10.1101/gr.139758.112 Mauro VP, Chappell SA (2014) A critical analysis of codon optimization in human therapeutics...synonymous codon usage bias, and its potential applications. Nucl Acids Res 15:1281–1295 . https://doi.org... GluCl ion channel genes for mammalian cells dramatically improves expression levels. Journal of Neuroscience...synthetic? How Addgene;s dataset reveals trends in biological innovation Resources on Addgene.org Check out...

...described above showed successful target modification, indicating that AAV is a good delivery vehicle for...overall packaging capacity but necessitating purification and co-infection of two AAVs. Cas9 orthologs...post-injection. Livers from these mice were histologically normal and liver injury markers were not increased...the AAV-SaCas9-sgRNA constructs mediate genome modification, but they did so without a substantial immune...enough to deliver with AAV. This system fuses catalytically dead dCas13b to the catalytic domain of RNA ...finding smaller Cas9 orthologs works for some applications, it doesn’t eliminate the need to deliver larger...

...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,...

...window onto communication between the muscle and brain, which may have therapeutic implications, say Hyo ...metabolites that are dynamically secreted by muscle during exercise to meet the brain’s physiological demands,” ...protects the brain from depression. Mice that are genetically modified to produce extra PGC-1a1 only in their..., the brain is protected from stress-induced biochemical changes that could otherwise lead to depression...

...speak for you (and you for them) Communication Being able to communicate effectively is part of what makes...excellent Technical knowledge and creativity Become a “thought leader,” develop a technical “niche,” develop...productive. Take all the opportunities you can to be communicating about science with others. If opportunities ... (be on a speaker committee) Follow industry publications like FierceBiotech to “learn the business” Attend...

...clones, it’s wise to quickly assess whether a significant number of cells have been edited. For many experiments...enzyme screening for resistivity to digestion, indicating targeted edits.   Interested in this method...sequencing confirmation. All knock-ins A method applicable to knock-ins both large and small is a software...consider using a version of Cas9 that has been specifically engineered for reduced off target editing, such...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...Resources at Addgene: Read our CRISPRGuide Get practical help from our CRISPR Protocols Find gRNA Design...

...which to test the validity of your biological model and the technical performance of your assay. But, in... a more-typical scale of tissue culture while still offering the potential for true biological insight...components, the model system and the assay are just as critical for success. Sometimes, you might have a few positive...facilitating more rapid follow-up. We hope the biological community will find these reagents to be useful...

...al. "Systematic identification of signaling pathways with potential to confer anticancer drug resistance... therapies,” said Kris Wood of Duke University Medical Center. “While we do not yet have a comprehensive... 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... he said. “It could [be used to screen for] morphological features, migration, or survival in different...

...Thousands of mutations have been implicated in cancer. Yet, the majority of them remain uncharacterized...annotate genetic perturbations that may play significant roles in cancer pathogenesis. Their work culminated... of mutations they identified included gene amplifications and fusions, missense mutations, and indels...mutations were found to be “neutral,” having no significant effect on cell viability compared to a wild-type...Approximately ~22% of the mutations, however, significantly increased cell viability, and these “activators...aberration drivers of pancreatic cancer." Nature communications 7 (2016): 10500. PubMed PMID: 26806015. PubMed...

...with the gRNA), application, cas9 species, PubMed ID, and depositor. The CRISPR applications we currently...shown to work in CRISPR experiments. Addgene’s dedication to resource sharing has led us to develop a searchable...validated gRNA Target Sequence Table. Disclaimer: The efficacy of gRNAs is impacted by the target location, target...the carats in the datatable headers to sort alphabetically or in ascending or descending order, in the...and final experimental outcome in the original publication. As you develop and confirm new gRNAs, please...

...Since the first research applications of GFP were published in the 1990s, biologists have spent a lot... mammalian cells, and it may find wide use in biological research. Photoconvertible fluorescent proteins...color was observed after excitation at 488 nm, indicating that spontaneous conversion did not occur. Tobacco... Sattarzadeh et al., Sci Rep. 2015 CC BY 4.0 Applications and limitations of GFP photoconversion One concern...2):131-3.  PubMed. Find plasmids from this publication at Addgene. Subach OM, Patterson GH, Ting LM...nmeth.1664. PubMed. Find plasmids from this publication at Addgene.   Addgene’s Fluorescent Protein...

...scientific community support open sharing. But, one critical part was missing when the COVID-19 plasmids first...Carolina at Chapel Hill University of Illinois Chicago                     Boston Children's Hospital                           ...                           National Institute for Physiological Sciences Broad Institute                                       ...                                       Chinese Academy of Medical Sciences University of Texas at Austin University of California...part of research projects, another way is to systematically offer thousands of constructs for tagged and...enables research on solute carriers (SLCs) for medical research Recently, the Michael Davidson Fluorescent...

... QC/QA team as a DNA Sequencing Intern. The application process When I first saw the comprehensive job...immediately clear that Addgene wanted to ensure applicants understood what the expectations were for the...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...team and shows how they fit into the system. Specifically as a co-op, it is encouraged to explore different...currently working on projects for the Science Communication function at Addgene. Working a 9-5 As it turned...