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...to autophagy-inducing conditions. Rosella makes it easier to study the mechanisms behind autophagy by... cells. Autophagy (Greek for “self-eating”) is induced by a lack of nutrients and targets cytosol and ... via proteasome degradation. Autophagy is then induced and quantified by measuring the amount of radioactivity...radioactivity in the culture supernatant (this is indicative of protein degradation). To control for protein...cytosol in an inactive form unless autophagy is induced. Then non-selective macroautophagy of the cytosol...some conditions it aggregates in an autophagy-independent manner. Additionally, tracking Atg8p or LC3 doesn...fluorescence. After 4 hours of nitrogen starvation to induce autophagy, red but not green fluorescence accumulates...

...You need a lot of time to let you mind process things. Sometimes finding that uninterrupted time to be creative..., but started looking for a new job because “it kinda sucked to work remotely. I was also tired of the...integrated and part of a team.” It took her two years to find her current position at Fred Hutch because her skill...through the tough assignments, the skills that Susan  finds important for her work include: “Listening. Evaluating...advice for getting started in science communication: “Find as many opportunities as you can to get your name...unpaid work as a student. For post-grad school individuals, Susan recommends starting a blog, but with one...internship. Application information can be found here Find tips for using social media as a scientist here ...

...channels, the team will be able to validate their findings in mammalian cells with both human and murine ...cytokines actually get out of keratinocytes and induce their effector functions. It’s an important process... Hospital Zurich. IL-36 cytokines are a potent inducer of an autoinflammatory loop: keratinocytes both...Lin got me interested in TS when I was a newly independent faculty member studying sex differences in thoracic...thoracic aortic disease,” Prakash says. In induced pluripotent stem cells derived endothelial cells from...thirty GNB1 missense variants identified from 58 individuals with GNB1 disorder since the identification of...allow us to enhance our biosensor repository as we find ourselves lacking several crucial biosensors that...

...version, but if you find yourself doing your own conjugation, it is important to keep in mind that you will ... be useful. Mainly because, well, antibodies are kind of hard to see. To solve this issue, researchers...antibody conjugates in an earlier post, where you can find some additional tips for working with these tools...affinity for avidin and streptavidin, both of which can bind up to four biotin molecules and can themselves be...with significant multiplexing potential. You will find antibody-oligonucleotide conjugates used in assays...protein-protein interactions with other peptides, like an Fc-binding domain (Figure 1).  Figure 1: Examples of...). If some of those lysines are in the antigen binding region or even just nearby, then conjugation could...

...skills, individuals also tend to have their own specialties. This type of team can exist indefinitely. If ... - you just want the necessary skills. You might find that your next paper is going to require help from...project that never ends is a poorly planned project indeed. Mission team A Mission Team is assembled to make...this year, I joined Addgene’s Green Team, a like-minded group of co-workers who want to make our organization...creating and strengthening those social bonds. So never mind how much better your work and productivity will ...someone to decide, someone to delegate, etc. If you find that there is not someone (or someones) taking on...easily discover you need to add some people if you find there’s a need for an expertise your current team...

..., coughing, etc. Also, a constant in-flow of individuals wherever the cell cultures are kept will increase...Contamination! Tissue culture media often have indicators included in the media that turns yellow from ...trigger a series of chemical reactions and this induces a noticeable color change. This method has high...fluorescent signals produced by these bacteria can hinder correct result interpretation. Also, the signals...Also, prolonged use of a particular antibiotic can induce resistance for other bacteria apart from mycoplasma... should be cleaned regularly and dampness of any kind should be avoided. Distilled water provided to maintain...because you are not the first person to face this kind of problem. Educate others when you can and help...

...that absorb photons for energy conversion or the induction of intra- or intercellular signaling pathways ...extra-chromosomally long-term expression and low ability to induce innate immune responses (Fenno et al., 2011). However...site-specific recombinases such as Cre or Flp that induce DNA recombination at defined recognition sites ...between mismatched recognition sites, its presence induces first opsin inversion, and then lox sites excision...opsin gene; in Cre expressing cells, however, Cre induces the recombination of the double-floxed opsin construct...FLEx-based vectors to study the neural circuitry behind the mechanisms of sleep and anesthesia. In Cre ...that it is possible to separate neuronal inputs to induce aversion- or reward-related behaviors. The high...

...preliminary data to establish your own lab and get an independent investigator grant (e.g. an R01 from the NIH,...makes time for you, as needed, this is a strong indicator that this person cares about your science and ...the reagents and outcomes. This is preferable to finding out you've been doing something all wrong or that... as they can result in spurious yet intriguing findings that fail to replicate. Plan experiments logically...great discoveries in science arose from surprising findings when studying a seemingly unrelated problem. However...As you get better at telling your story, you can find clearer ways to consolidate or present your data...you give your talk or poster to enough different kinds of audiences and if you pay attention to how the...

...standardized validation techniques for academia, industry, and especially the clinic. Popular validation...enhancing confidence and reproducibility of a study’s findings. Table 1: Biased Genotyping Options Technique... Figure 1: Schematic representations of the indicated unbiased genotyping options. Figure reproduced...experiments Unbiased detection methods are excellent for finding evidence of DSBs throughout the genome. However...due to >3 bp mismatches or that are sequence-independent are rare, but they are detectable using just ... et al. "High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells." Nature...high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing." BMC...

...allows for tagging and visualization of specific, individual proteins under endogenous regulatory control....your own gene-tagging constructs below. Read on to find out how these plasmids work and to discover more...cells so they can go to work. Figure 3: Inducing the double strand break and introducing the tag...BioRender.com. Plasmids, plasmids everywhere To find out more about gene edited hiPS cell lines generated...Catalog on the Allen Cell Explorer, where you can also find the certificate of analysis provided with each cell... epitope tags (e.g. FLAG and STREP) and auxin-inducible degrons (AID). Further, the Yamamoto PITCh system...on microhomology-mediated end-joining (MMEJ). To find out more about gene edited hiPS cell lines generated...

...species can be indicated in the “Species Other” column. Relevant Mutations Indicate the amino acid...reagent/antibody, unspecified, or other. If other, indicate type in “Vector Type Other” column. For example...cloning, gene synthesis, Gibson cloning, ligation-independent cloning, restriction enzyme, TOPO cloning, unknown...or room temperature. Growth Strain Please indicate which growth strain Addgene should propagate your...in any of these standard strains, choose other, indicate strain in the “Growth Strain Other” field, and...2024.    Additional Resources on the Addgene Blog Find Articles Citing Addgene Plasmids Searchable and ...CRISPR Experiment Suggest A Plasmid You'd Like to Find at Addgene Resources on Addgene.org Start Your...

...phosphopeptide binding domain FHA. The FHA1 domain binds to phosphorylated PKA substrate, inducing a conformational...deposits and we hope they'll make it easier for you to find and use the plasmids you need. If you'd ever like...undergoes a conformational change that permits it to bind to the CIB1 (cryptochrome-interacting basic-helix-loop-helix... by measuring growth of the ΔguaB pDCAF3 strain. Find the decaffeination plasmid here. Quandt et al. ...adapt similar FLINC biosensors to work in your lab. Find the FLINC biosensors here. Mo et al. Nat Methods... extinction coefficient when compared to sREACH. Find the FLIM-FRET acceptors here. See Addgene's FRET...

...with a common dilemma. Request the vectors you can find and use them as-is, saving time and effort but risking...available for request on the Addgene website, both as individual plasmids and as the Fragmid kit. You can also...timeline of the Golden Gate (GG) cloning approach. Individual modules and a sample ligation are depicted. (... sequencing is just part of doing good science!” Find the Fragmid kit here!  Cloning specifics Fragmid...Pol 2 protomer, Cas protein, ect...). Whatever individual components you choose will assemble in the expected...lab tested the Fragmid system by sequencing 60 individual assemblies created over five months. When they...one plasmid was the correct sequence for every individual assembly. What destination vectors are available...

...the hopes and concerns of these communities. This kind of engagement and two-way dialogue is crucial if...meetings of experts and leaders from academia, industry, community, education and government; advising...make their own decisions about genetics, both as individual consumers of technologies and as societal participants...three years. pgEd is a small team comprised of individuals from diverse backgrounds and disciplines, and...is to liaise with different organizations and individuals, to identify ways of collaborating, and to organize...academic venues such as scientific, educational or industry conferences, as well as schools, community events...and in helping you decide whether you enjoy this kind of work. It is also beneficial to hone your non-...

... Resources on Addgene.org Find mammalian plasmids for your research Find viral vectors for infecting... hood contamination free, but you should keep in mind that UV light only kills microorganisms that it ...results. Most cell culture media contain the pH indicator phenol red (pH range 6.0 (yellow) - 8.0 (red))...Anything between orange and light orange usually indicates that the cells have grown enough, are making their...interested in Glycobiology and Immunology. You can find Sana on LinkedIn.   References 1. Yin, R., et al...Gene expression profiling analysis of bisphenol A-induced perturbation in biological processes in ER-negative...Pagliara, Patrizia, et al. "Ostreopsis cf. ovata induces cytoskeletal disorganization, apoptosis, and gene...

...through the use of constitutive and inducible promoters. Inducible promoters are extremely useful if a...including their advantages and caveats to keep in mind before choosing a system. Mammalian expression systems...bacterial cell. Transformed cells propagate, are induced to produce your protein of interest, and then lysed..., under the control of the T7 lac promoter and induced by lactose, provides the highest level of protein...recombinant proteins under the control of the galactose inducible promoter (GAL). Other commonly used promoters ...promoters include the phosphate and copper inducible PHO5 and CUP1 promoters respectively. Yeast cells are grown...exudates Resources on the Addgene Blog Learn about inducible promoters Learn about repressible promoters Check...

...for its chance in the spotlight — Cas3. Cas3 can induce genomic alterations on a scale that other Cas effectors...capabilities of Cas3 and its partner in crime, Cascade. Find Cascade-Cas3 plasmids in Addgene's collection! A...Regardless of the subtype, the mechanism of Cascade binding and subsequent DNA degradation appears to be conserved...ensures that there isn’t a template strand left behind that the cell can use to repair. Figure ... DNA degradation continued. However, Cas3 cannot bind DNA on its own and needs to remain anchored to Cascade...throughout the process. The reeling of ssDNA and unwinding of dsDNA occur simultaneously, which creates a...reeling by the Cascade-Cas3 complex in nucleotide unwinding steps. Molecular Cell, 70(3), 385-394.e3. https...

...sample for further downstream applications. As individual libraries can vary significantly, it’s important... an amplification protocol developed for each individual pooled library; generalized amplification protocols... you need 30 to 200 times as many colonies as individual plasmids in your library. As an example, if you...  Pro tip! Since you probably do not want to individually count 500,000 colonies, you’ll likely rely on... DNA, you will likely need to perform several individual transformations, such as splitting 400 ng of ...of DNA in 100 μL of cells over four individual electroporations. The specifics of this can be based on ...limiting your growth time (e.g., 16-18 hours). Finding reference protocols There are many decisions to...

...Libraries Find More CRISPR Resources at Addgene: Find CRISPR Plasmids Read our CRISPR Guide Find tools to...additional experiments investigating the role of individual genes or entire pathways in a particular disease...specific gene within the genome (see Fig. 2). Individual gRNAs are designed in silico using publicly available...Addgene, with more being added all the time. Keep in mind that libraries are only as good as the experiments...genes that confer drug resistance when mutated. Findings from this type of experiment can shed light on...phenotype. Each hit from the screen should be independently validated to ensure that the desired modification...

...software could also be used as a simple tool to rapidly find one or a few gRNAs suggested to be effective by ...knocked out at the population level, but each individual cell only has a maximum of one gene knocked out... an approach, you lose the unbiased potential to find things you are not looking for, but there are also...important for their function. Their migration is induced by various types of interactions with their environment...approved drugs already exist. If you are lucky and find that a druggable gene has a central role in your...eleven published libraries are embedded. These were kindly donated by six different research constellations...contain multiple gRNAs targeting all genes in the indicated species and were suggested to be the best gRNAs...

...you can read more about the Addgenie behind the blog post and find more of their work. Articles from guest...collection of material so that you can more easily find what you’re looking for. Whether you’re joining ...cover topics outside of these five areas and you can find what you need by field (ex: microbiology, neuroscience...publishing, under the Other dropdown menu. You can also find information on using Addgene’s website, the latest...Now, you can search for the articles you want to find without even leaving the Addgene blog! Just click...well as their topic category. New author pages to find articles from your favorite bloggers Like a blog...

...experiments in labs around the world were put on hold indefinitely due to COVID-19. In our lab, we were interested...placing orders for paper x on date t. We cannot use individual plasmid orders, because when a PI or lab places...several plasmids at once, breaking the statistical independence assumption of the Poisson process. This modification... by COVID-19, so we defined the COVID-19 event windows as 01/15/20 -- 03/15/20 for China and 03/11/20 ... The inferred parameters suggest that COVID-19 induced a 2x–5x reduction in the rate of scientists placing...more dramatic than the COVID-19 effect, though we remind the reader that this is not a causal interpretation... In conclusion, our model infers that COVID-19 induced a 2x–5x reduction in the rate of scientists placing...

...you’ve thought about starting a journal club is to find a topic and seeing if others are interested. You...run the journal club. Some are led by a single individual who walks through the paper figure by figure....single presenter summarizing the paper’s major findings and then focusing more on broader implications...implications rather than diving deep into the individual data points. All formats should be an open forum where ... attend for scheduling conflicts. This helped us find a time that accommodates more people. The feedback...very point is discussion. Perhaps the project is winding down. Perhaps other projects have taken on a larger... often driven by one or two participants. If you find yourself in this situation, considering stopping...

...it lacks Cas9’s other HNH endonuclease domain, indicating that Cpf1 functions differently. Since Cpf1 loci...red) or LbCpf1 (blue). Mismatch positions are indicated below each graph. Figure from Kleinstiver et al... and LbCpf1 ribonucleoproteins (RNPs) failed to induce off-target editing in a cell culture model. The...CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227. Find plasmids from this publication at Addgene. 2. Zetsche...Biotechnol. 35.1 (2016): 31-34. PubMed PMID: 27918548. Find plasmids from this publication at Addgene. 3. Kim.... Nat Biotechnol. (2016). PubMed PMID: 27272384 Find plasmids from this publication at Addgene. Kleinstiver... Nat Biotechnol. (2016). PubMed PMID: 27347757. Find plasmids from this publication at Addgene. Makarova...

...Erhardt S, Lindskog M, Ruas JL. doi: 10.1016/j.cell.2014.07.051. PMID: 25259918 Muscle over mind. Moon HY...transgenic mice that helps to explain how it works. The findings in a recent issue of Cell demonstrate how PGC-...PGC-1a1, a transcriptional coactivator induced in skeletal muscle by exercise and endurance, protects the...in the body, the brain is protected from stress-induced biochemical changes that could otherwise lead to...of the kidney or the liver.” The discovery is a window onto communication between the muscle and brain...kynurenine metabolism and mediates resilience to stress-induced depression. Agudelo LZ, Femenía T, Orhan F, Porsmyr-Palmertz...

...Welch says, “[It] seems counter-intuitive, but I find office work more mentally challenging than lab work... job” could entail any number of career paths--industry, nonprofit organization, law office, journal editor...your time. As my colleague Mike Lemieux told me, “I find that at a desk, it is much easier to stick to timelines...who coordinates cross-team projects at Addgene, I find that my vocabulary improves on a weekly basis. ...amazing thing to be a part of and really helps me get behind a project or initiative.” If you think the meetings...science in the first place. Finally, if you still find the idea of not holding a pipette again upsetting...the community (and sometimes conduct their own independent research!). There are also organizations like...

...Resources on Addgene.org Find protocols of molecular biology techniques Find plasmids at Addgene Make ...2017 RSpace was developed with these principles in mind, and in response to further input provided by the...repositories. Below I describe how each of these kinds of connectivity facilitates reproducibility. Connectivity...Slack conversations and import them into RSpace. The kind of informal conversations about research that can...enables users to conveniently select and deposit individual documents or more complex datasets into the desired... edit to particular groups and/or particular individuals, the issue of third-party access to the ELN arises...rory_macneil.     Additional resources on the Addgene blog Find tips on choosing an electronic lab notebook Watch...

...cancer. These later stages of a cancer diagnosis indicate, for Stage 3, that the cancer is large in size...size and may have spread while Stage 4 indicates that the cancer has spread from where it originated to ...if not all) of the cancer cells that may be left behind after surgery, while being less traumatic to the...into the cytoplasm, migrates out of the cell, and binds cells that can begin an immune response. This important...and even toys! Contrary to the belief of many individuals, asbestos has not been banned in the United States...such a ban, and the president of the organization, Linda Reinstein – whose husband passed away from mesothelioma...Alliance. Additional Resources on the Addgene Blog Find out More About Cancer and Addgene's Cancer Collection...

...PUF Assembly Kit makes it possible to devise RNA binding proteins to hit any target of interest. The new... to human proteins known as Pumilio/fem-3 mRNA binding factors (PUF). In a single step, researchers can...domains for RNA specificity engineering. “The RNA binding domain is interesting because by changing certain...proteins include eight modular repeats, each of which binds a consecutive base in its RNA target. By changing... acids, it’s possible to produce a protein that binds an alternate sequence. By comparison, Abil explained...explained, the specificity of other RNA binding domains depends on the secondary structure of RNA. As a result... are endless.” Abil says the new tool might even find therapeutic application in addition to their use...

...researchers are on the line. With these priorities in mind, many researchers go to great lengths to keep accurate...efficiently accomplish our daily goals. These tools remind us of our daily tasks, make our days more effective...effective, in both academic circles as well as in industry. The news is good. In their ideal form, ELNs provide...budgets, “a large percentage of survey respondents indicated that cost was a significant barrier to ELN adoption...to digital for example): “… 99% of respondents indicated that ease of use would influence their ELN choice... of it all in paper notebooks. Following these findings, we defined the priorities for future ELN development...schedule online meetings with the support teams behind them.  Conclusion Researchers spend 40% of their...

... are equal. Created with BioRender.com. This kind of normalization is a nearly ubiquitous tool, but... assumptions that their loading controls are independent of these other sources of error. It’s important...housekeeping protein in your context. If you don’t find anything, you’ll need to conduct a control experiment...strongly influenced by unexpected variations in any individual protein. This ensures you are truly comparing...with β-actin (1:2000). Representative of three independent blots. B–C) Graphs showing the relative intensity...increasing concentration of β-actin in panel A. Indeed, the quantification in panel B is not especially...total protein are compared. While a higher slope indicates higher sensitivity, linearity is the most important...

...expression under both rhamnose- and arabinose-inducible promoters. Tight control of toxin expression is...leading to counter-selection escape). The rhamnose induction system is well-characterized and quite tight (...placed the toxin genes under control of a rhamnose-inducible promoter, though Khetrapal et al. also demonstrated...that the arabinose promoter worked too. Rhamnose induction relies on the transcriptional activator rhaS. ... copy of rhaS with a strong synthetic ribosome binding site (using the online calculator from the Salis...are real! Planning an allelic exchange experiment? Find the pTOX plasmids here! Continued development of...the Addgene blog Read our Plasmids 101 blog posts Find our CRISPR blog posts Learn more about Lambda Red...

...Expression Kit FREQ Seq PUF Assembly Kit Below you’ll find information about what these kits contain, how they...Biological Chemistry 290.8 (2015): 4728-4740. You can find more info on pCri in Mary Gearing’s blog post on...translation initiation sequence known as the Species-Independent Translational Sequence (SITS). This sequence ...composed of repeat segments that can be engineered to bind specific RNA bases. By swapping out these different...repeat segments, you can create a PUF protein that binds your RNA sequence of interest. The Zhao lab has ...contains all the variant PUF segments required to bind any 8 base RNA sequence. The segments are provided.... Using this system, you can create custom RNA binding proteins in as little as 3 days. Abil et al., 2014...

...to be binding at particular genomic sites when in fact they are just non-specific or indirectly interacting...is the go-to method for mapping where a protein binds genome-wide, and has been widely applied in many..., an enzyme-based protocol for identifying DNA binding patterns that overcomes some of the limitations...Protein A fused MNase is then added and Protein A binds Immunoglobulin G (IgG) thus targeting MNase to antibody...provides an excellent additional measure of DNA binding sites in combination with existing ChIP-seq data...with CAPTURE Additional Resources on Addgene.org Find simple lab protocols Browse CRISPR plasmids for ...

... PhDs Making the Leap to Industry." He successfully transitioned to industry after his PhD and a couple...mental freedom. Practical approaches Engaging in mindful meditation, taking field trips, and spending time...gain fresh perspectives. Such activities foster a mindset that embraces the unexpected and encourages exploration...Serendipity is not just a stroke of luck; it's a mindset that can lead to unexpected discoveries and innovative...successful research career, whether within academia or industry. Albert Einstein once said: “There are only two...serendipity into your research but also cultivate a mindset that is adaptable, creative, and poised for groundbreaking...

...s-level job postings on a job search website like Indeed. Do the job descriptions sound interesting? Is ...work something you can see yourself enjoying?  To find master’s-level postings, try adding “master’s” to...filters to limit results to people that work in industries you’re interested in (i.e. biotechnology, pharmaceuticals...switch your field of work, both in academia and in industry. Advancing along a career path in these two fields...fields looks different however. In industry the career ladder is larger and there are more opportunities...Career advancement for PhDs, both in academia and industry, often means moving from the lab to a desk, although...on Addgene.org Check out Addgene's job openings Find eBooks from Addgene, including the Science Career...

...-4 colonies to ensure that you find the correct product. Once you find a colony with the appropriately...Posts Resources on Addgene.org Find Empty Vectors for Your Research Find Additional Protocols Browse Our... Some of these include, TA cloning, ligation independent cloning, TOPO cloning, one step cloning, and ...Li, et al., in 2011. This method is ligation independent, it does not require purification of insert or...simplified, purification-free, sequence-and ligation-independent PCR cloning method." BMC biotechnology 11.1 (...

...feedback when it is merited. Surprisingly, some people find this hard so get used to it by making it a concrete...However, I have also found that scientists seem to find it easier to give feedback in formal sessions using... Here are the 3 things that worked least well. Find out the Effect of the Feedback: How does this feedback...over, what would you do differently? Beware of indirect language You must be honest about performance ... mistakes I see is managers who hint at or use indirect language to talk about real performance issues...issues. Watch this little video to learn more about indirect language from Steven Pinker. As a manager you ...and clear. There is place in human discourse for indirect language, but it is not the way to go when giving...

...single peak, indicating one amplicon and no primer dimers. (b) A shoulder peak indicating primer dimers...the right conditions and parameters, some people find it can detect even smaller differences, and it can...dimers. (c) A double peak indicating 2 amplicons. Image credits (respectively): Zuzanna K. Filutowska; Selma...different types of pipettes and pipetting methods to find what’s most consistent in your hands.  When using...target in the reaction should be compared to an individually run sample when developing the protocol and ...and optimized until the multiplexed and individual values match across the board. If the samples or sample... e-book on applying MIQE guidelines! With these kinds of resources, you will soon become a qPCR master...

...short-hand for Clustered Regularly Interspaced Short Palindromic Repeats, in Pubmed returned 728 articles (3/12...because the Cas9 nickase doesn't have the ability to induce double-stranded breaks like the wildtype Cas9 does...'G'. Q4: Where are double strand breaks (DSBs) induced, compared to where the target site sequence (protospacer... colonies, then genotype them, it is possible to find both single-allelic and bi-allelic cells. Single-allelic...double nickase system, with PX335 vector, you need to find two guides for each cleavage site, but this system... we usually pick 3–6 guides around the region to find the most efficient guide (most guides would work...single-cell colonies, grow them up, then perform individual genotyping (using Sanger sequencing, for example...