... donor and acceptor fluorophores, it’s best to consider these cases separately when talking about the ...the tools that are available. Let’s consider the donor first where labeling specificity is most important...AnapRS Mammalian Cells Note: This is not an extensive list Acceptor labeling: Sometimes it's okay to... (i.e. >100 Å). Researchers have developed an extensive array of chemical labeling strategies suitable...understanding of your protein that can lead to novel insights about its biology and its behavior. See the table...covalent labeling of recombinant protein molecules inside live cells." Science281.5374 (1998): 269-272. PubMed...

...from these sites. The Zhang lab generated a comprehensive library of each unique TF open reading frame...cellular fates were then matched with candidate TFs responsible for that differentiation.   Fig. 2: Single...Previously engineered TF libraries were not as comprehensive as this one, so it is an invaluable tool to ... the 24 TFs they tested, 22 expressed markers consistent with the cell type that was predicted based on... if the differentiated cells had morphologies consistent with the predicted cell type (e.g., axon-like...

...conformational changes lead to an increase in fluorescence intensity from the inserted GFP. After characterizing iGluSnFR...by glutamate, and tweaking it to improve its responsiveness, they showed that iGluSnFR could detect neuronal...and produces stronger fluorescent signals upon sensing glutamate in mouse and ferret models. Furthermore...recently made iGluSnFR3 variants that are more sensitive and better localized for synaptic glutamate response... and produce stronger fluorescent signals upon sensing glutamate using both 1-photon and 2-photon imaging...

...can be engineered to be cell type specific. Once inside the host cell, the virus unleashes its cargo – ...signals which can traffic their viral particles inside the nucleus, without the need for envelope disassembly...viruses do. They are generally used to achieve transient, high expression of the relevant cargo without...Capacity (>8kb) High Transduction Efficiency Transient Gene Expression High Immunogenecity AAV ...Immunogenicity Infects Non-Dividing Cells Transient Gene Expression Low Tropism for Mouse Cells and...

...DNA packed into our chromosomes is very much 3-dimensional, as you can see by this post’s chromatin illustrations...PIs, articles, genes, and blog posts are not comprehensive, so just because you do not see a name or gene...the gene you’re looking for on this list, please consider searching directly on our website. In-exhaustive...Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents. Yamagata M, Sanes...chromatin and the epigenome. If you have been considering some new techniques, the posts below may steer...

...personalized steps like “check drawers for light-sensitive reagents” or “re-sterilize now.” Have a physical...Soon you’ll figure out what prompts you need to consistently and accurately run a protocol.   Figure...experiment than it is to rerun an experiment, so consider your prep an investment in accuracy.  Bench setup...hasn't. Developing simple habits like these can consistently reduce errors as you work, and the possibilities... to a protocol if you’re in a hurry, but let’s consider it this way: When you’re stressed or rushing, ...

.... The next insight was to assemble a synthetic transcription activation complex consisting of multiple...transcriptional activation of endogenous genes and it consists of three components: 1. A nucleolytically inactive...-mediated screens exhibited a high degree of consistency and validation with >80% effectively enriched...optimize humans for CRISPRi and CRISPRa.” Before considering the use of pooled libraries in your experiments...

...They all found the support of the group to be intrinsic to their success. Their stories made it clear ...programs. The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet together... of people who are all interested in making a transition. Schedule your first meeting and get organized...in a rotation. The leader for that meeting is responsible for choosing a topic and leading the discussion... your successes and support each other as you transition to the next stage in your careers! Stay in touch...

...channelrhodopsin used as a sensitive fluorescent voltage indicator with near-complete desensitization in continuous... the imbalance of electrical charge between the inside and the outside of the cell. This imbalance is ...Q239 - A GEVI based on the fusion of the voltage sensing domain of a Ciona intestinalis voltage sensor and...anion channel. It is triggered by a short low-intensity blue light pulse and stays open for a longer duration...

...secondary considerations to validation, application specificity, and experimental considerations - no other...has been validated in your specific application. Consider also that there may be variations within an application... your needs.    Multiplex experiments Finally, consider whether you will be using other antibodies in ...from a vendor that you use frequently, with a responsive and knowledgeable technical support team, is ...

...spectral properties of the fluorophores you are considering to make sure that they’ll be compatible with ...antibody conjugates (especially if you start to consider clinical applications). But there are a few other... at least mention. Oligonucleotides allow for sensitive detection of low abundance targets with significant...mechanisms. Most take advantage of chemical groups intrinsically present on antibodies, such as the primary amine...can conjugate an antibody yourself allows you to consider a broader selection of antibodies for your experiments...

...proteins: a relatively pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive GFP variant, super ecliptic...Rosella is a pH-sensitive fluorescent biosensor that was recently deposited with Addgene by Dr. Mark ... and recycling. The key to Rosella’s autophagy-sensing abilities is that its fluorescence emission spectra...regardless of its localization in the cell. SEP’s pH sensitivity means Rosella will fluoresce green unless it’...

... learned over the years with the hope that our insights can help steer you in the right direction when... of this is gospel, but we think it's worth consideration. Everything will go wrong Whenever an order ...easily as needed. In this case, the physical considerations of the laboratory make object composition a...technical details of the problem you're solving, but consistency is key. Some namespace collisions you may encounter...treating these pieces of data in ways that are inconsistent with how they are being treated in the lab, ...

...given problem, Empowers the writer, And offers insight into a career as a Professor. Step 1: Your ...sections in a NIH RFA, for example, and tips to consider while writing each section. Research challenge...time to review the literature and offer educated insight into what the desirable features of future technologies...deem your proposal successful. Typical proposals consist of 3 to 5 aims, with secondary milestones to meet... the application. Make sure to invest time and consider the reviewer's comments. Whether the proposal ...

...virion is a non-enveloped particle of ~100 nm, consisting of a capsid surrounding an inner core which contains...adenovirus: the virion is ~ 100nm in size, and consists of an icosahedral capsid made of 3 types of proteins... for creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the ...purification of rAdV uses cesium chloride (CsCl) density gradients combined with ultracentrifugation to ... rAdV genomes/mL) can be measured by: Optical density: particle concentration is measured by looking ...

...Interactions Difficult to detect weak and/or transient PPIs. Many PPI identification techniques rely ...often prevents the identification of weak and/or transient interacting proteins. No spatiotemporal control...mass spec analysis. Biotinylated proteins are considered possible interaction partners if they are enriched...BioID, is also useful for identifying weak and/or transient protein-protein interactions since the biotin ...cell lysis and lysate fractionation. Important considerations for Split-BioID It’s important to remember ...

...some reliable controls. 2. Viral stocks may be sensitive to freeze thaw cycles. Depending on the researcher...ultracentrifugation of the viral stock, followed by resuspension of the collected particles in a smaller volume...Davis et al., 2002). However, polybrene is highly sensitive to freeze-thaw cycles and should be stored in ...enhance transduction efficiency in cells that are sensitive to the toxicity of polybrene (e.g., hematopoietic...PMID: 12050007. 7. Stockschläder, M., et al. "Expansion and fibronectin-enhanced retroviral transduction...

...III), each responsible for transcribing different types of RNA: Polymerase I is responsible for ribosomal... Rho-independent termination is also known as intrinsic termination, and it relies on the formation of...ribosomal RNA, Polymerase II is responsible for mRNA and miRNAs, and Polymerase III transcribes tRNA and other...polymerase-associated proteins, CPSF and CstF, which are responsible for recruiting the cleavage and polyadenylation...

...injections are most often used when injecting a cell suspension to create xenograft models. Restraint: back scruffing...sounds very disruptive, but surprisingly, it is considered minimally invasive. Restraint: anesthesia Location...common. Due to the intubation, this procedure is considered quite invasive. Restraint: anesthesia Location...introduction to common injection routes in mice! When considering what method you’d like to use, remember that ...animals: routes of administration and factors to consider. PubMed, 50(5), 600–613. https://pubmed.ncbi.nlm.nih.gov...

...to their high density, which can be collected afterwards. You can then remove the density gradient material...certain in vitro and in vivo applications, you can consider using unpurified AAV from the supernatant (Goodwin...cells and supernatant then need to be purified by density gradient centrifugation (e.g., iodixanol) using...assay for adeno-associated virus vectors with sensitivity sufficient to detect single infectious events...

...this temperature and can bind to the primers.  Extension- The temperature is then raised slightly to Taq...  This process of denaturation, annealing and extension is repeated 25-35 times to exponentially replicate...droplet PCR or ddPCR is a method that provides ultrasensitive and absolute nucleotide concentration, unlike...more complicated to develop and is often less sensitive than PCRs that use a single pair of primers, like...Diagnostics NGS library prep QC ddPCR Determine ultrasensitive and absolute nucleotide concentration of starting...

...because they are inside the virus itself. While these impurities are generally considered innocuous for ...helper plasmids or the cell genome can be packaged inside the vectors during production. These impurities...NGS at Addgene to sequence all the DNA packaged inside the viral particles (Figure 1). Briefly, packaged...contamination is a good start, but to determine the sensitivity of our method we wanted to see how low we could...

...that onto a mass spec. That's very time intensive and expensive. There's the promise that we could instead...DNA systems capable of storing data and computing inside living cells. He's also a co-founder of the BioBricks...describing the development of “transcriptors,” transistor-like digital genetic switches that enable cellular...proteins. That's central to everything that goes on inside a cell. Until we get a handle on precise, forward...

...protein combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal...(Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and nMagHigh1...able to reach deeper tissues. Additionally, high intensity exposure can cause DNA damage. Far-red light, ...Michael Lin, who developed a chimeric protein consisting of Cas9 and a photo-dissociable dimeric green... anti-CRISPR Acr protein, AcrIIA4, to a photo-sensitive LOV2 domain from A. sativa (Bubeck et al., 2018...editing For more details, check out this comprehensive review comparing optogenetic CRISPR techniques...

...immune system directly). It may seem odder when you consider that out of the 6 x 106 virus-like particles that...DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell” (or host cell...reverse transcriptase are packaged (Wu et al., 1997) inside of the capsid . This is useful for viral reproduction... particle, some of the RT protein gets packaged inside of the vector particle, along with its RT-deficient... natural virus, this new chromosomal locus is considered “proviral DNA” (Rowe et al., 1971) and the full...

... venture presents itself even outside of work, consider challenging yourself, take the opportunity, and... all the things for which you are officially responsible on paper, and take some time to write down all... have to exercise in order to manage these responsibilities. If you are in charge of completing many ...management and organizational skills. If you are responsible for working with lots of students, managing inventories...the short list of transferrable skills here and consider which of these skills you’ve used in your own ...

...use a standard curve and provide some general considerations for scientists planning to use them in their...to 1:32. Each individual step of the series is consistent at 1:2. When preparing a dilution series, use...users aim for an R2 above 0.95.    Additional considerations When running assays that rely on a standard...not fall within this range, it may be time to reconsider your graphing program. Summary Few things in ...run.  When setting up your dilution series, use consistent dilution factors, change pipette tips regularly...

...of current new and exciting literature. As I transitioned out of academia and away from the bench I certainly... figure organization with a different person responsible for each figure. Others still are more casual...everyone to offer thoughts you may not have even considered. These questions should be tailored to the group... not actively participating, it may be time to consider ending the meeting. This is a common failing in...participants. If you find yourself in this situation, considering stopping (temporarily or permanently) the recurring...

...are some potential complications that must be considered when using this method:     Cells must be amenable...to directly deliver a ribonucleoprotein (RNP), consisting of the Cas9 protein in complex with a targeting...gRNA from a commercial vendor, this can often be expensive. An alternative approach is to express and purify...is most appropriate for a given experiment. Considerations for RNP-mediated CRISPR experiments While using...with these types of experiments. One important consideration is that the Cas9-gRNA RNP will be degraded by...

...immunoprecipitation (locus-specific ChIP). These methods consist of insertional chromatin immunoprecipitation (iChIP...Advisory Board.  Addgene: Your lab has worked extensively with enChIP systems. Can you describe this technology...technologies. The locus-specific ChIP technologies consisting of iChIP and enChIP are recent examples. We also...community is a kind of duty for researchers when considering the peer-review system of journals and grant ...Identification of Proteins Associated with an IFNgamma-Responsive Promoter by a Retroviral Expression System for...

...and it will teach them to respect their own responsibilities. Another tip: don’t miss your grandfather’... the evening, or in a few years) when life responsibilities are less You can work at home sometimes, but... the burden alone You, alone, are not solely responsible for care and attention to your home and family...doctor without your help. Your partner is equally responsible and capable. The only exception is that men cannot...tasks – try to resist the impulse to control by insisting things always be done your way. Be patient with...

...viral vector solution. Since this technique is intensive and requires an electron microscope, it is not...image shows that the vast majority of the vectors consist of full particles (white arrowhead) relative to...from different sources in the same experiment, consider retitering both AAV in your lab. The absolute ...does not give an indication of infectious titer, consider titrating each lot of AAV in vivo to determine...Additional Resources on the Addgene Blog Important Considerations When Using AAVs Addgene's AAV Quality Control...

... package stays in transit for a certain number of days and tracking average transit time for each courier...having some materials available to order, yet not consistently accessible, wasn’t an option. So we identified...For example, when we began offering temperature-sensitive materials, we noticed a large percentage of these...distributor allowed us to ensure that temperature-sensitive materials like viral preps and libraries were ...

...emerged from intensive engagement with a wide range of people at the University of Wisconsin in 2011/12 ...reproducibility is limited in two ways – only data inside the ELN is reproducible, and only the highly restricted...principles emerging from the engagement with the Wisconsin research community were to: (1) prevent vendor... choosing an ELN for your research, be sure to consider how its features do and don’t support reproducibility...

...Specificity concerns? If off-target effects are a worry, consider a dual-sgRNA system. This system uses a “nicking...little homology to other regions of the genome and consider using a Cas9 variant with higher specificity (...requirements, giving it more flexibility. A consideration with this tool is that the desired edit will...activators The simplest of the dCas9 activators consists of dCas9 fused to a single transcriptional activation...Mediator complex, generally the strongest activator, consists of a dCas9-VP64 fusion and a modified gRNA that...

...AAV delivered genes. AAV serotypes have been extensively studied in order to define those that target ...weakly expressed. As these epitopes tags have no intrinsic visual signal, they cannot be detected directly...neurons. As compared to epitopes tags, FPs are intrinsically fluorescent and can therefore be used in live...darkened chromophores. These probes are both intrinsically fluorescent and can be detected with highly ...tags like GFP. Specifically for STORM, the high intensity signal provided by smFPs can result in better ...

...resistant to antibacterial drugs. Resistance can be intrinsic, meaning a basic characteristic that naturally...resistant somehow overtime. One fantastic example of intrinsic resistance would be Mycoplasma species’ resistance...possess cell walls. As a result, Mycoplasma are intrinsically resistant to this class of antibiotic. Acquired...be unharmed by the compound it produces (think intrinsic resistance), but surrounding microbes would be...prescribed to them This may seem counter-intuitive considering the other three scenarios, but a short treatment...

...sure your cells have first reached an appropriate density. Cells in the exponential growth phase are alive... prevent the culture from reaching the desired density within a reasonable time-frame. For these cases... growth media, or if your plasmid is low-copy, consider reducing the antibiotic concentration. Growth ...accomplished by growing your culture to the desired density at 37°C and reducing the temperature or moving ...TB or 2XYT is optimal because of the high cell-density they support; however, minimal media supplemented...

...antibodies, increasing the fluorescence signal intensity. However, you need to be careful when selecting...interest is not located on the cell membrane but inside the cell. Due to their chemical nature, antibodies...nucleus intact. If markers of interest are located inside the nucleus, a different fix/perm kit is required...that can enter cells freely are typically used. Inside the cell, the ester group is cleaved off by enzymes... yielding a stepwise decrease in fluorescence intensity in a (histogram) flow plot (Figure 4b). Through...

...metabolic engineering.  The E. coli genome has an extensive research history and characterization. Many proteins...cardiovascular disease, specifically stroke and hypertension (Iannaccone et al., 2009). Rodents have also...amphibians in science with early research providing insight into some of the most significant and fundamental... plant model organisms in the world. With its extensive scientific history, highly characterized genome...Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428, 493–521 ...

...concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2, 4) ...vector with Gibson assembly. While this is a comprehensive mutagenesis method, a drawback to using Gibson...scanning libraries. PFunkel: Of prior existing comprehensive mutagenesis methods, PFunkel offers the best...PCR-based approach for generating a customizable comprehensive mutagenesis library that’s ready to be tested...Elad, and Marc Ostermeier. “PFunkel: Efficient, Expansive, User-Defined Mutagenesis.” Ed. D. Dafydd Jones...

...gene of interest) and prevent incorporation of extensive sequences used for cloning into the final translated...Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium... integrity, expression level, and aggregation propensity in four expression systems: E. coli, wheat germ...frequencies across timepoints or populations. This kit consists of a 48 plasmid adapter library, with each plasmid...Morgan’s blog post on the PUF Assembly Kit for some insights on its potential from its creators. We hope you...

...included inducible Cre, such as the tamoxifen-responsive CreERT, the coupling of Cre-lox with FLP-FRT ...limited due to low resolution or were very time-intensive, necessitating the development of a new technique...This system differs from Brainbow-1.0 in that transient Cre recombination is required to prevent constant...tissue was challenging due to low fluorescence intensity, caused partially by fluorophore photoinstability... the Brainbow technique will provide important insights into the complicated physical organization of ...

...prokaryotic minions that carried out the labor-intensive process of replicating your new plasmid: the Escherichia...certain restriction enzymes that are methylation sensitive. dnaJ Mutation in a chaperonin gene Increases...strains are all based on E. coli K-12 and are considered BSL-1. Table 2: Lab strains of E. coli Strain...Dam or Dcm methylated, allows for methylation sensitive restriction enzymes to cut the plasmid after preparation...tables are by no means exhaustive! For a more comprehensive list and additional information, visit OpenWetWare's...

...gRNAs! Comic by Maya Kostman.   Important considerations before you start an experiment with CRISPR ...expression. Location and sequence are important considerations for designing your gRNAs. For indels, it's ...off targets. For CRISPRa and CRISPRi, these considerations are of roughly equal importance (target should..., 2020). is not limited to single nucleotide transitions but still requires a nearby PAM, although these...The off-target activity of gRNAs is important to consider. While the basic landscape of mismatches that ...

...FLEx Vectors Important Considerations When Using AAVs Three Key Considerations For Precise Neuronal Targeting...-target expression.  Some classes of neurons transiently express TH during development or have low levels...titer to prevent off-target expression of the sensitive FLEx-split TVA receptor used for tracing neural...recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors. Proc Natl Acad Sci USA 116:27001...

...the Moloney Murine leukemia virus (MoMLV) and consisted of two plasmids: the packaging plasmid containing...vectors Although retroviruses are still in use, considerable efforts have been devoted to develop efficient...generation. The 1st generation of lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector... from one batch to another. For experimental consistency it’s a good idea to purify and titer your virus...sucrose to the supernatant to create a gradient of density that favours virus precipitation. My tip for this...

...for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...cooperate to cause a higher-energy electronic transition in a fluorescent molecule (see figure to the ...technical specificities have to be taken into consideration especially in the use of fluorescent proteins...wavelength can significantly affect emission intensities from fluorescent proteins (Salomonnson et al....Marcello, et al. "Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial...

...before you even receive the product? How can we consider the environmental impact of those processes? Who...laboratory products that emphasizes Accountability, Consistency, and Transparency. This label scores products...might sound risky in terms of contamination, but consider flow cytometers. These fluidic machines handle...through an EPA-approved process and no longer considered hazardous. Then, they use this material to produce...

...goes beyond such restrictions, you may want to consider a systemic capsid for your AAV.   Systemic capsids... definition of systemic capsids. After some consideration, we offer this definition: A systemic capsid...capsids are engineered, as well as highlight some considerations for their use. Then, we’ll provide information...and/or its intermediate engineered precursor. Considering systemic capsids for your research? While systemic...payload — there are a few additional, specific considerations you’ll need to be aware of before starting ...choice on which systemic capsid to use. Factors to consider include the delivery method, mouse strain, target...recommend you check out Caltech’s CLOVER Center’s extensive repository of published systemic AAV experiments...