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...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...

...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...

... 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...

...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...