...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...

...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...

...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...

...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...

... 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...

...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...

...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...

...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...

...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 ...

...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...

...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 ...

...elements often determines the type of plasmid and dictates how it might be used in the lab. Below are some...common plasmid types: Cloning Plasmids - Used to facilitate the cloning of DNA fragments. Cloning vectors... gene function as well as identify changes or mutations that may cause certain diseases. Sequencing DNA...

...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...