...and disease states, their circadian regulation, as well as in important physiological states such as adipogenesis...which then return information such as nutritional status. Within the animal kingdom, this information is...that interact directly with NRs) exist in steady-state complexes with secondary coregulator (or co-coregulator...whole complex.  For example, the phosphorylation status of SRC-3 defines its association with other members...

... PE6e-g editors with mutations to the Cas9 domain but found that these mutations only improved efficiency...created and evaluated 19 PE1 variants with RT mutations known to increase activity, enhance binding between...additional nuclear localization sequences; and two mutations in Cas9 previously shown to improve nuclease activity...effective prime editor proteins, but found that the mutations that emerged depended strongly on the target edit... ~ ~ ~ ✓ ✓ ✓ R221K N394K Cas9 mutations Improve Cas9 nuclease activity ~ ~ ~ ...

...antigens not present on the cell surface. The presentation of antigens can also be affected by cell fixation...quantified using the Attune NxT Flow Cytometer with representative images showing the staining intensity in the... Correctly interpreting your data therefore necessitates careful design of RNAi sequences, confirmation...validation is critical to ensuring accurate interpretation of your results. Therefore, taking advantage...Uniprot ID P21579) for use in western blot, immunoprecipitation, immunofluorescence and flow cytometry. F1000Research..., Robertson, J., & McPherson, P. S. (2019). Implementation of an antibody characterization procedure and...

...poster, you shouldn’t hesitate to be creative. After all, your poster is a representation of your work and ...most prominently, making it the star of your presentation. Portrait. This style displays a poster vertically...Morrison, a psychology doctoral student at Michigan State University. The idea is that the main research finding... center of the poster in a large font. Hence, spectators can read the text from many feet away and immediately...

...components of the plasmids to partial, full, or annotated plasmid sequences. These sequences can also be...the deposited sequence(s) in SnapGene. SnapGene annotates the plasmid maps based on SnapGene’s “Common Feature... function of the plasmid. This includes point mutations, frame shifts, missing elements, and nucleotide...plasmid that are less likely to affect function when mutated; elements that are difficult to sequence; approaches...Sequence discrepancies are not always a result of mutations in the plasmid, but can arise due to errors in...

...range of motion, a commutator allows the patch cable to connect to the laser and rotate 360° without a loss...wavelength to stimulate your opsin. Each opsin has an excitation spectrum (use the resources discussed in post...variant you wish to use to determine the ideal excitation). Lasers can be purchased through Laserglow, ...channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive, open, closed/light insensitive...

...effects this mutation has on locomotion in our computer model to the effects of this mutation in the real...knowledge sharing, in the form of workshops, presentations, and lectures. There are currently around 1,...to modify the worm genome and make additional mutations in our worms.  We are on the lookout for genome...contributes each month - with only occasional supplementation with grant funds, money is always tight. Of...

...from New England Biolabs. Golden gate assembly limitations Embraced by the synthetic biology community, ...or two-step assemblies. We have reduced these limitations through the use of engineered Type IIS restriction...shown above) are flanked by BsaI sites in an orientation that results in 5’ four base overhangs after ...transformants onto the selection plate. Figure 2 shows representative transformation plates obtained from 1-, 12-...standard reaction conditions. This data set allows quantitation of sequence-dependent ligation efficiency and...

...Addgene makes this easy by providing a template citation for you to use in your Materials and Methods and...Addgene catalog number to easily find template citations for Addgene plasmids you have used in your research...depositor has indicated that there is a particular mutation found within the gene/insert, this information...questions about our plasmid pages, please don't hesitate to contact us at [email protected] Resources on... on the Addgene Blog Read about Our New Citations Feature Check out Our Walkthrough of the Deposit Process...

...expression? Are you trying to create a single point mutation? Do you want to create a fusion with a reporter...generating knockouts or knock-ins, or introducing mutations and tags, while a catalytically dead dCas9 can... Base editors can help you make precise point mutations near the gRNA target site. Choose an appropriate...repair (HDR) template to create knock-ins, point mutations, or to add a tag, you will also need to deliver... the expression system you choose in many ways dictates the best method for introducing those CRISPR components...

...chemical and peptide-based proteosome inhibitors, glutathione S-transferase (GST), which can be fused to recombinant...recombinant proteins for one-step purification with glutathione, can also protect against proteolysis. That’s... then your first step towards detecting, immunoprecipitating, or purifying that protein may be to fuse...Purification and Stability Good for purification with glutathione; protects against proteolysis, but may reduce...column runs. The TEV protease itself, with various mutations used to increase its stability activity, can be...

...characteristics and capabilities an ELN needs to facilitate true reproducibility. The following are critical...making it easy to get data out of the ELN; and (2) facilitate inter-operability with other research tools and...describe how each of these kinds of connectivity facilitates reproducibility. Connectivity to other data sources...original formatting. In this way data is moved from a static and isolated vehicle and becomes part of the dynamic...Framework, Data Dryad, and Figshare. Any ELN that facilitates reproducibility has to make it easy to export...

...population and track connections to that population. Limitations of common genetically encoded neuronal tracers...attached to a protein of interest (POI), that facilitate immunohistochemistry (IHC) experiments with tag-specific... A new and improved neuronal tracer With the limitations of epitope tags and fluorescent proteins, it ...track with these classic probes. To overcome the limitations of existing FP and epitope tags, researchers ...Bobby Henderson, a physics graduate from Oregon State University. Find the Spaghetti Monster probes at...

...methods: Quantitative PCR (qPCR) Digital droplet PCR (ddPCR) Historically, a quantitative DNA hybridization...to report that over 95% of viral capsids in representative viral vector preparations are genome-containing...1. Fagone, Paolo, et al. "Systemic errors in quantitative polymerase chain reaction titration of self-...High-throughput droplet digital PCR system for absolute quantitation of DNA copy number." Analytical chemistry 83.22...

... to master ddPCR.  Finally, it’s a good idea to state why you’re applying to the specific company or lab...rationale for designing it the way you did, and state that it would help you manage a large number of ...yourself and your background. It’s a great place to state why you’re applying to the job, company, and/or ... talked about it yet, it's also a good place to state why you are interested in working for this lab and...informal poll of Addgene hiring managers. All of them stated that a good cover letter, one that explained how...

...transfer’. Resistance can also arise from random mutations in the genetic sequence that happen to be beneficial... if some didn’t develop, either through random mutation or horizontal gene transfer, means to defend themselves... the only method of determining drug resistance status, it is a very common, well-trusted method that ...smarter, not harder. We can utilize tactics like rotating antibiotic treatments, so that exposure to each...effective drug targets (ones that are less likely to mutate or change in order for the bacterium to survive...

...life science literature is a litany of false interpretation in cell culture for this reason. Unreproducible...Required for Acquisition of Anoikis Resistance and Metastatic Potential in Adenoid Cystic Carcinoma, Up-Regulation..., papers are retracted and there is a loss of reputation that can impede collaborations, especially with...between cells lines, as this can be dependent on the mutation rate of the cell line, but it is broadly recommended...2010. 2. Gold, Michael. "A conspiracy of cells." State University of New York Press, Albany (1986). 3. ...

...non-fluorescent, and misfolded FP-fusions on quantitative imaging experiments and cell physiology in general...population is not readily apparent and can confound quantitative imaging experiments or even negatively impact...misfolded dark FP fusions including erroneous quantitation of fusion protein levels. It remains unclear...fluorescent proteins and were re-engineered by mutating cysteine residues and N-glycosylation consensus...proteins. Our recent publication illustrated the quantitative increase in fluorescent signal achieved when...

...distance between the two fluorophores and their orientation to one another can affect FRET. When it comes...between the two fluorophores and their spatial orientation will be fixed. The quantum yield of the donor...between the two fluorophores is linked to their excitation spectrum overlap. If you choose a pair too close...increased. This method is straightforward and quantitative, but it is destructive and cannot be used for...method is also less sensitive to direct acceptor excitation artifacts and it is possible to use a non-fluorescent...

...devised means of co-opting phage genetic systems to facilitate homologous recombination in bacteria. These quirks...non-edited cells to survive have inactivating mutations in the Cas9 or spacer sequence, and these rare...pneumoniae and can be used to generate multiple mutations simultaneously (Jiang et al. 2013). Genome editing...recombineering, with 96-99% efficiency for point mutations and 66% efficiency for small insertions. Multiplexing... E. coli, the system was used successfully in Tatumella citrea, another species of Enterobacteriaceae,...they were effectively able to introduce point mutations in P. aeruginosa, Pseudomonas putida, Pseudomonas...

...diversity in fluorescence brightness, intensity, excitation and emission spectra. The lab of Dimitri Deheyn...group to perform independent two-color optical excitation of neurons. The paper describes five previously...variety of contexts. In order to overcome these limitations, they evolved and characterized an improved peroxidase...Clonetegration could become a “valuable technique facilitating genetic engineering with difficult-to-clone ... sites that flank a DNA module in an inverse orientation, so that multiple DNA components can directionally...synaptic partners) technique based on functional complementation between two non-fluorescent split GFP fragments...combinations (depending on the number of copies). This facilitates the distinction of neighboring cells in imaging...

...conditions. As a facultative aerobe, it can thrive with or without oxygen. Additionally, its status as a Biosafety...used as a model organism for over 60 years with a status as the most studied prokaryotic organism. With ... biofilms, and much more. Because of it’s BSL-1 status, E. coli can be used as a model to relate these...plant species and metabolic properties affecting adaptations.  Arabidopsis thaliana flowers. Image from...

...different mutations found in leukemia, the Lowe lab was able to identify a set of mutations that made ...Lowe’s lab utilized mice to study how different mutations in leukemia impact different treatment regimens...by a specific gene. For example, several genes mutated in human cancers have a homolog involved in yeast... yeast: A model organism for studying somatic mutations and cancer." Nature Education 1.1 (2008): 183....

...genetically encoded fluorescent probes. One of the limitations is the size of GFP: ~240 amino acids or about...supplementary figure C). However, these assays are qualitative. The Organized Smooth Endoplasmic Reticulum (...the appearance of OSER whorls. This assay is quantitative and is currently the standard for assessing ...straight fusions, without any linker. When both orientations are available, a direct comparison of these ...PMCID: PMC3324619. Cranfill, Paula J., et al. "Quantitative assessment of fluorescent proteins." Nature ...

...in cells, they have various disadvantages and limitations – for example, microtubule-destabilizing drugs...damage other cellular structures, and genetic mutations are not easily reversible. Late last year, the... et al., 2022), that overcomes many of those limitations (Figure 1). Figure 1: Opto-katanin induces fast...reversible microtubule severing and has very low dark-state activity and toxicity. All of that means this light-powered...moderately-enhanced 10x and 30x SuperPLDs avoid dark-state activity and cytotoxicity better than the 50x and...

...also use different cytoskeletal machineries to facilitate transport: retrograde transport relies on dynein...2014). It is currently unclear which proteins facilitate transport of neuronal tracing compounds and this...-Gold (Schmued and Fallon, 1986) are used. To facilitate cellular uptake of these dyes in vivo, microinjection...a negative impact on cellular health. Another limitation of traditional tracing compounds is that their...rAAV2-retro’s altered amino acid sequence might facilitate interaction with uptake or intracellular transport...mechanisms of intracellular transport of AAV could facilitate the development of more powerful AAV serotypes...

...genome and are inefficient at introducing point mutations. In 2016, the researchers at the Broad Institute... envisioned a new strategy to introduce point mutations in the genome. They fused a catalytically impaired...a tRNA deaminase enzyme, TadA) to induce a A→G mutation at the target genomic loci. Together, CBE and ...the prime editor can install all possible point mutations, delete short stretches of DNA and even insert...doi.org/10.1038/nprot.2013.143 Seligman LM (2002) Mutations altering the cleavage specificity of a homing ...

...need representatives and policymakers who not only believe in the separation of church and state, but ...Institute, most women of reproductive age live in a state with at least two abortion restrictions that conflict...are that we, as a society, need to: 1) use more citations for out data so our sources are readily available...the Vancouver SciCATs. Follow her on twitter @Point_Mutation, and @SciCATSyvr. Additional Resources on ...

... aside for the event. As guests arrived, some gravitated towards the sushi and drinks, while others headed...hour, we feasted on sliders, hot dogs and a mashed potato bar (complete with all the fixings), as well as...ending the celebration. The party later moved to State Park, where I had a great (maybe too great!) time...last 10 years @Addgene Check out our impressive stats from 10 Years of Plasmid Sharing Or read more about...

...final version of the document. Set and manage expectations When the researchers involved in a project are... crucial that everyone meets early on to set expectations for the output that will come from the collaboration...should then hold each other accountable to these expectations once they’re set by checking in about them at...speed up the future directions of the project. The state of research is constantly shifting, and long-distance...

... does have limitations. The enzyme-based CUT&RUN method overcomes many of these limitations and makes ...graduate student at UNC Chapel Hill. Chromatin immunoprecipitation followed by high-throughput sequencing, ChIP-Seq... binding patterns that overcomes some of the limitations of ChIP-seq. CUT&RUN, which stands for ‘Cleavage...

...several struggles with these techniques: unwanted mutations due to excessive PCR cycles or low fidelity Taq...purify and sequence the plasmid to confirm no mutations were introduced during PCR or any subsequent steps...fidelity DNA polymerases, I have never had any mutations introduced into my inserts. However, with any ...technique there is a chance of introducing an unwanted mutation. Tips for high efficiency cloning Now that you...

... used SMRT sequencing, TA-based ligation and tagmentation-based methods for AAV genome sequencing (Tai...self-complementary AAVs and the TA-based ligation and tagmentation preparation approaches described previously ...single-stranded and both TA-based ligation and tagmentation require a double-stranded template.  Using viral...the mix to serve as effective substrates for tagmentation-based library preparation procedures. This finding...

...choice while bypassing the intermediate pluripotent state. Though a valuable tool for regenerative medicine...least day 13. However, distinct transcriptional states were seen as early as day 6. All together these...stochastic, but rather there’s a particular cell state that’s permissive to reprogramming.In this lineage...reprogramming. MettI7a1 encodes an uncharacterized putative methyltransferase. Adding Mettl7a1 to the standard...

...however, has inherent limitations because it turns a dynamic process into a static measurement and it provides... acidic environments at 586 nm. Regardless of excitation wavelength, Keima emits at 620 nm. The ratio ... Noda, Takeshi, and Daniel J. Klionsky. "The quantitative Pho8Δ60 assay of nonspecific autophagy." Methods...

...single molecules and newly formed proteins.  Limitations with GFP in imaging  While GFP has been an immensely...to study proteins in vivo, it does have some limitations. GFP has long protein maturation times which ...antibody based probes a solution? To address these limitations, scientists have co-opted antibodies for tagging... the National Academy of Sciences of the United States of America, 69(9), 2659–2662. PubMed PMID:4560694...

... sketch on the board without leaving Zoom. The annotate function can also make the screen-sharing experience...top of the screen and every participant get to annotate things as they come up in their minds. The final...writing and this recent article published in PLOS Computational Biology (Frassl et al., 2018): “Science is increasingly...where all lab members talk no science. No data presentation will appear here, and only general chatter will...

...train and acquire the data you need on the new instrumentation. Bonus reasons Visiting gives you access to...This probably isn’t the lab’s first request for visitation if they have specialized skills and equipment...so that you can work together to ensure your expectations are reasonable, your reagents are onsite before...include, but is not limited to temporary housing, transportation, living expenses, and necessary reagents. Your...

...forming concatemers later. The "FS" stands for Fragmentation System and this is the most important part of...for library preparation using this kit is: Fragmentation and end repair Adapter Ligation Clean up/Size... the start of the workshop, NEB gave a brief presentation describing the technology. Then we split into...and network with other technicians/company representatives. I got to talk with a technician from Neon ...

... gRNA representation, produce high titer lentivirus from the library, ensure gRNA representation in the...technologies in plasmid form and are proud to have facilitated their fast adoption. However, in many cases the...screening-scale experiment achieving the gRNA representation recommended in the publications associated ...

...success. It is imperative that you resist the temptation to “just do it myself” or micromanage –you don...develop the web page. The team manager must then facilitate appropriate communication between members of ...only way and may not even be the best way. This facilitates success and trust. Micromanaging is bad for everyone..., meeting minutes and plan changes. Written documentation helps make sure everyone is on the same page...

...samples, the rest will be put into a status we call “Archived.” This status allows us to store plasmids and..., please fill out this form and we’ll add your citation to the plasmid page on our website. Contributing... contain the expected features and are free of mutation.  Once the samples arrive, each group of plates...

... used FRET, but it eliminates the need for an excitation light source. Instead, a luciferase substrate...complement optogenetic tools, where light used for excitation can cause unintended activation of the optogenetic...various colors. In fluorescent imaging, you use the excitation wavelength to specify which FP emission you’ll...Nano-lantern system does not use external light for excitation, you instead have to rely on differences in emission...

...effective manager. Mismanaging (no pun intended) expectations —yours and your team's—can lead to frustration...unnatural for those accustomed to hands-on work. The temptation to "do it yourself" can result in burnout and...Miscommunication often arises when managers fail to set expectations or provide timely feedback, leading to unproductive...discussions to understand their perspectives and facilitated a team meeting to highlight their complementary...

...collaborating as they craft sentences for abstracts and presentation slides, learn from audiences as they do science...something they did not understand about your presentation, question whether other aspects of the work ...primary focus When writing a paper or preparing a presentation, it's easy to forget that you have a much bigger...practice.  Ask them what they learned from your presentation and evaluate whether or not you’ve succeeded...

... methods of manipulation are genetic knockout mutations and treatment with small molecules/drugs. With...with a uniform population possessing a specific mutation or phenotype. For example, a drug screen may be... performed on a cell line possessing a patient mutation to assess how patients may respond to drug treatment...survive as these must have acquired a favorable mutation. But if the cellular pathway of interest conveys...

...for NHEJ repair regardless of end structure, and dictate the major events of the pathway: Broken ends are...processing of DNA ends tends to be the point where mutations are introduced. Repair of Cas9-induced breaks ...about a two-thirds chance of causing a frameshift mutation. Of some importance, the deletion can be less ...double nickase” strategy vastly reduces breaks and mutations at off-target sites. Christina Mork contributed...

...variation of gene expression. Because pSC101 is not representative of all types of plasmids, the stabilized promoters... number. During the shift from exponential to stationary phase, the number of plasmids per cell increases... plasmid copy number during the transition to stationary phase, Segall-Shapiro et al. placed a trans-acting... such as qPCR or microarray. But, aside from facilitating the joys of a p-value < 0.05, having such fine...

...times, LinkedIn may revoke your ability to send invitations. Finally, if you don’t know clearly why you are... box, it will send an unpersonalized, generic invitation. Don’t try to connect from the LinkedIn phone... app either as this also results in a generic invitation. 3) Connect with people you have really talked... with this and as long as you personalize the invitation with your intention for connecting, this is probably...

...understanding of how our bodies work, new treatments for debilitating diseases, and maybe even ways to alleviate health... broaden the conversations about genetics and facilitate dialogue between scientists and communities. ...With our focus on engaging the community and facilitating dialogue, a major part of our everyday work ...jargon-filled language in academic writing and presentations. But for many people who don’t work in these...