We narrowed to 1,015 results for: LEA

...first experience, I learned the details of grant writing, volunteered to take the lead on various applications... Thus, every single sentence and figure must be clear, concise, meaningful, and supportive of the mission...funded proposal will address these limitations and lead to new milestones in the field. Research strategy...that the goal and the impact of the project are clearly stated and align with the description of the RFA...see what tips they found useful. Step 4: Read and learn from the reviewers Reviewers may take 6-12 months...application. Although some comments may make it clear that the reviewer misunderstood the point that was... being made, take it as an opportunity to make clearer statements on the next round of applications. ...

...conditions, the protease cleaves itself and the degron off at the protease cleavage sequence. The tag is ...function, the SMASh tag self-cleaves itself when you want the protein around, leaving a minimal additional sequence...asunaprevir, the viral protease can no longer self-cleave and the protease and degron remain attached to ...protein-of-interest and the CRBN E3 ligase complex leading to ubiquitination and proteasomal degradation. ...HaloTag is quite large (33 kDa) and does not self-cleave off of the protein like the SMASh tag does. However...round of evolution (AID-2) to reduce some of the leakiness associated with the system, but the general concept...

...stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss how NgAgo is faring with...regions of the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering...mammalian cells and one which diminishes the endonuclease activity of the enzyme. In vivo, only nascent...be rapidly degraded by the cellular machinery, leaving a small amount of active NgAgo/ssDNA complex in...manages to form a functional complex with ssDNA leading to inefficient downstream editing. Overall, the...suggestions and tips with #NgAgo The Addgene Blog - Leave suggestions and tips in the comments section below...someone came up with a mutant NgAgo (or a novel endonuclease) protein that can form a complex with a 5’-P-ssDNA...

...will be to your colleagues. If you ever have any questions about our plasmid pages, please don't hesitate...best use of all the information provided by your colleagues. Top of the plasmid page The ‘breadcrumb’ trail... is listed on the plasmid page.  The paper link leads to a page listing all the plasmids from this paper...References sections when citing the use of your colleague’s plasmid. Use the Addgene catalog number to easily...which experimental systems it was validated in.   Please keep in mind that all of the information on our...Check out Our Walkthrough of the Deposit Process Learn How We Do Quality Control at Addgene Resources ...Resources on Addgene.org Join the Addgene Community Learn the Benefits of Depositing Browse the Repository   ...

...classification system for the array of CRISPR technologies. Learning the general principles of this classification ... Cas3. Cas3 functions as both a helicase and a nuclease, unwinding and cutting up DNA after recruitment...systems are also employed as CRISPR transposases, by leaving out Cas3. Type I is further subdivided into subtypes...and DNA, though it is largely believed that the cleavage of DNA is the primary function of this type of...consider to be an integral component of CRISPR — a nuclease effector. However, IV-C does in fact have a helicase...caused by Cas12 create short 3’ overhangs, which leads to slightly increased efficiency when conducting...and binds the RNA target before initiating RNA cleavage. Created with BioRender.com.   What’s your...

...for Science Learn How Future of Research is Advocating for Change Within the Sciences Learn How Changing...partisanship, including more scientists in our leadership, and raising awareness regarding the pitfalls... Nicholas Thompson is a PhD Candidate studying Nuclear Science and Engineering at Rensselaer Polytechnic...women of reproductive age live in a state with at least two abortion restrictions that conflict with scientific...local law enforcement, and, although there was at least one person vocally protesting us, he was not threatening...I marched to advocate for the process we use to learn about the world. In a way, I marched for truth. ...that I loved - there were booths for children to learn little things about science. We need to keep encouraging...

...C-terminal tags for mTagBFP, TagRFPt, EGFP, mVenus, mCerulean3, mKOFP2) and empty vectors that have standard...expression from these polycistronic cassettes could lead to comparable experimental results. Using PiggyBac...monocistronic expression of either variation of Klf4 did not lead to the observed reprogramming discrepancies. The...Listen to our mScarlet Podcast Segment    Photocleavable protein PhoCl expands optogenetics toolbox ... mechanism - the PhoCl protein is irreversibly cleaved when exposed to light. Robert Campbell’s lab illustrates...repository. These variants can be used as tools to learn about the influence of structure on an FP’s properties...results argue that transcription activation by SOX2 leads to its reprogramming effects. Plasmids from the ...

...bacteria to release the plasmid, separate the cell debris from the plasmid, and then clean-up the plasmid...the plasmid DNA itself, which can happen if you leave the sample in a lysis buffer for too long or if ...plasmid DNA from unwanted cellular “debris”; and then clean-up the plasmid DNA (Figure 2). For most kits, the...DNA. Image created in BioRender.com. For the clean-up stage, plasmid purification kits typically use...approaches involve binding the plasmid DNA from your cleared lysate to a substrate, washing away left-over debris...the columns can be “clogged” by excess material, leading to less DNA in your final eluate. Bead-based kits...blood, virus, or other samples, for PCR and gel cleanup, and for extracting RNA! (That’s a blog for another...

...Everything I know about virus, I learned from Addgene,” or “I learned to clone from the Addgene website...country. In China, direct shipments weren’t able to clear import control. Partnering with a distributor allowed...repository by making it easier to find materials and learn about them. As early as 2007, we began grouping ...their lab, in their experiments, to enable virtual learning during the pandemic. Feedback like that lets us... not done yet… in fact, we may never be! We’ve learned that increased accessibility is achieved through...Through updating and expanding our website, we’ve learned about the importance of smaller, systemic changes...to easier, faster, more reproducible science and learn what Addgene can do to help. More resources on ...

...viral vector for our service; leave a comment; or take our AAV survey. Learn more about GCaMPs and other...editor of Addgene’s eBooks — including the recently released Viral Vectors 101 2nd edition — I was incredibly...in 2016. We realized we could apply the lessons learned from our plasmid lab to develop a reliable and ...It could be a matter of timing, as jGCaMP7 was released relatively soon after GCaMP6. It could be a matter... vector service. But in the years since, we’ve learned so much about what it takes to make these viral... Vectors 101 eBook and blog posts. The lessons learned from this process have shaped and supported our...growth and development. We’ve shared what we’ve learned, through manuscripts (Ersing et al., 2023; Haery...

...soon. To request other nucleases or other features and functionalities, please email [email protected] by Jessica Roginsky, Scientific Support Lead at Synthego. Article source: Step-by-Step Guide ...read the paper on bioRxiv.   Using the ICE tool Learning how to use the ICE software tool is quick and ...that need optimizing and no complicated steps to learn. For increased flexibility and scalability, the ...Protospacer Adjacent Motif (PAM) sequence for the nuclease used. Currently, ICE is configured for Cas9 fromStreptococcus...guaranteed editing with the CRISPR Gene Knockout Kit. Please contact us at [email protected] if you have ...guest blogger, Jessica Roginsky. Jessica Roginsky leads the scientific support team at Synthego. Her work...

...improve nuclear localization of fluorescent proteins, they found that adding a triple nuclear localization...ever like to write about a recent plasmid deposit please sign up here.  Here's what you'll find in this ...TEV), a highly sequence-specific protease used to cleave affinity tags from purified recombinant proteins...techniques to introduce time modulation of the TEV cleavage and improve utility of the system for use with...localize to the right places, which can provide misleading information on where exactly a structure or protein...found that too high expression of these markers can lead to non-specific labeling. They generated plasmids...includes 95 plasmids consisting of CRISPR/Cas nucleases, base editors, gRNA backbones, and promoters for...

...thing. I experienced it, and many of my scientist colleagues have as well. Making the transition from the ...something. The thrill of having an experiment yield clear results and being the first, and for a very brief...family three times during a 10 minute conversation. Leaving a tight-knit team like that can and will be a little...world imagines when they think “scientist.” But leaving the bench does not mean you’re not a scientist ...culture shock above, but after polling some Addgene colleagues, we’ve come up with a list of things to consider... It’s a lot easier to manage your time. As my colleague Mike Lemieux told me, “I find that at a desk, ...non-scientists! Maybe you’ve just spent 5-10 years learning to “speak science,” and virtually every person...

...very difficult and very time consuming to treat. Learn How Antibiotic Resistance Genes Are Used in the ...susceptibility test (Figure 2), and requires at least 18-24 hours to obtain results. While this is not...our recent blog post on CRISPR antimicrobials to learn how scientists are using CRISPR to fight bacteria... schools, and households by observing proper cleanliness and personal hygiene standards. Handwashing is...essential component of preventing infection, as is cleaning and disinfecting frequently touched surfaces like...scary to think about, but we can all do our part to learn all we can, and minimize both our risk and the risk...Sheet  Additional Resources on the Addgene Blog Learn How Antibiotic Resistance Genes Are Used in the ...

...researchers and publishers. Nonetheless, the initial release caused a storm as the organization left open the...comprised of numerous objections and concerns. Perhaps least compelling of these were those from researchers ...unlikely. Usually, the originating scientist and colleagues are privileged in viewing their data months or...in reproducing experimental data are likely, at least in part, due to our inadequate cataloguing of how...endless types of data). Rather, PLOS appears to leave the scope of data to be shared to individual authors... intimidated by having to contact more senior colleagues. Secondly, science is highly technical and this...product. If we don’t take interest in how it is released, we will be in danger of having to deliver on ...

...addition to this cis cleavage, the authors showed that Cas14 could indiscriminately cleave ssDNA in trans like...variants share the presence of a predicted RuvC nuclease domain, characteristic of CRISPR-Cas enzymes. ...and more complex Cas9 and Cas12 proteins. Cas14 cleaves ssDNA But what is the function of this new system...bacteria strain. They found that Cas14 could bind and cleave a targeted sequence of ssDNA. Unlike Cas9, Cas14...for instance, the enzymes are activated and can cleave fluorescent probes of dsDNA and RNA in trans. Fluorescent...30337455. Additional resources on the Addgene Blog Learn more about DETECTR Browse all of the CRISPR blog...

...GreenGate are type IIS restriction endonucleases. They are like other endonucleases except that they cut at arbitrary...lab.” Introducing GreenGate As Lohmann and his colleagues describe in the PLoS ONE paper that introduced...be very careful with the type IIS restriction endonuclease of choice, BsaI, which can be very sensitive...to temperature fluctuations. He says their lab learned the hard way after the cloning method, which had... They finally traced the problem back to the endonuclease. “It’s just a sensitive protein,” he says. “...longer works, just trash it.” Lohmann and his colleagues including Joachim Forner now expect and hope ...

...is still important. It needs to express value, clearly communicate exactly what a site offers to their... for users to get where they desired. It became clear to us that we needed to improve search and navigation...as simplifying the page, and making it all look clean and easy to use.  Easily search for plasmids (and... new search bar is now front and center, with a clear call to action - Search! Along with this we’ve also...element. We also set out to make sure the page looked clean, professional, and modern. Instead of trying to ...next science challenge. If you have any feedback, please contact us and let us know how you’re feeling.  ....  Additional resources on the Addgene blog Learn more about Addgene's new search tool Read more about...

...enzymes. Note that when using the nuclear fix/perm buffer to label intranuclear proteins of interest, you can... ligases; and many more. This process, however, leaves the nucleus intact. If markers of interest are ...can label cytosolic proteins alongside the intranuclear ones. You do not need to use a separate fix/perm...typically used. Inside the cell, the ester group is cleaved off by enzymes, turning the resulting molecule ...You made it through this blog post and hopefully learned a few new methods of target visualization beyond...specific downstream applications, while maintaining a clean cell surface, dump gating is a useful way to get... labeling can be performed on cytosolic and/or nuclear markers. Lastly, stains, based not on antibodies...

...for you, if you can identify them in yourself and learn how to sell them. Types of Conflict Before explaining... on something and a disagreement festers, it can lead to hurt feelings, low morale, poor decision making...situation. If left unchecked, this type of conflict can lead to people disagreeing with each other just for the...the real source of the conflict. It can be easy to leap to the conclusion that a conflict is personal (”... listener, but I aspire to  be. If you don’t at least aspire to be a good listener and consciously practice...wanted. However, a poorly brokered compromise could leave everyone feeling vaguely unsatisfied. In arbitration...than either A or B, but the two parties will have learned that by working together, they can create unique...

..., because leaky expression of toxin could select for mutations in the toxin itself (leading to counter-selection...gene with an antibiotic resistance cassette. This leaves a “scar” in the chromosome, sometimes interfering...ways to create scarless mutations, ones that don’t leave any undesired scars in the chromosome. These methods... The resolution step (“double crossover”) which leads to the desired mutation and excision of the vector... paper. We selected the three toxins so that at least one might be useful in a broad array of pathogenic... switched over to pTOX. However, we feel it has clear advantages, and are hopeful that continued development...Plasmids 101 blog posts Find our CRISPR blog posts Learn more about Lambda Red recombineering Resources ...

...protein’s reading frame, leading to the production of a smaller, but at least partially functional protein...individual experiences progressive muscle wasting leading to death at around 30 years of age. Despite the...depositor Charles Gersbach used paired zinc finger nucleases to remove exon 51 in DMD patient myoblasts. They...force, and other muscle tests, muscle function is clearly improved, although not to wild-type levels. With...that dystrophin restoration is maintained for at least six months. Future directions and obstacles to clinical...through genomic excision of exon 51 by zinc finger nucleases. Mol Ther. 2015a Mar;23(3):523-32. PubMed PMID...PMC4393360. Additional Resources on the Addgene Blog Learn How CRISPR Could Be Used to Treat Cataracts and ...

...By mutating one of two Cas9 nuclease domains, researchers created the CRISPR nickase. Nickases create...mutation inactivates the RuvC domain, so this nickase cleaves only the target strand. Conversely, the H840A mutation... in the HNH domain creates a non-target strand-cleaving nickase. Instead of cutting both strands bluntly...distances (40 - 130 nt). From these results, it’s clear that editing is much higher in the PAM-out configuration... At the AAVS1 locus, the two nearest gRNAs had cleavage sites 12 nt and 13 nt away from the target site...wide margin, exhibiting >20% repair efficiency. Clearly, a nickase strategy can expand the targetable region...post. Additional Resources on the Addgene Blog Learn about Homology Directed Repair 3 Tips to Improve...

...depurination, the hydrolysis of the glycosidic bond leads to the release of a purine (adenine or guanine) from the...DNA are repaired by base excision repair, but can lead to mutations in single stranded DNA undergoing replication... from E. coli is the presence of the protein endonuclease I encoded by the gene endA. Nowadays, the most...gene has been removed/mutated to neutralize the nuclease activity. For strains that still carry this gene...introduced in the purification protocol to remove endonucleases activity. For these reasons choosing the correct...most downstream processing. On the other hand, nuclease free water is a great option because it provides...

...its endonuclease activity to generates a ssDNA substrate which is then nicked by its endonuclease domain...engineered Learn about the parts of the AAV transfer plasmid  Resources on Addgene.org Learn about AAV...of Rep78 and Rep68 contains a DNA-binding and endonuclease domains, all four Rep proteins share a common...virion assembly, the role of MAAP has not been clearly defined yet. Now you’ve got a basic understanding...AAV RepCap plasmid! Check out this blog post to learn more about the AAV transfer plasmid! References...

...Careers in Science Communication blog series, you’ll learn about Caitlin Runne-Janczy, a Product Development...While her love of educational science writing is clear, Caitlin’s career plans weren’t concrete when she...what I wanted to do, but knowing that I wanted to leave the bench.” Her path to figuring out her career ...writer.” At the end of her postdoc, Caitlin had a clearer idea of her career goals: “My plan was to go into...for part two of Caitlin’s interview where you can learn what it’s like to work for a science education company...series!  Additional Resources on the Addgene Blog Learn more about alternative science careers here Be like...while in grad school Resources on Addgene.org Learn more about careers at Addgene ...

...non-academic career Learn more about alternative science careers here Resources on Addgene.org Learn more about...series was written for selfish reasons: I wanted to learn about careers in science communication. When I started...away-from-the-bench career option. So if you’re interested in learning more about science communication careers, you’...was a science writer, reached out to me. She was leaving a writing position and looking for someone who ...s paid off. Work is never boring. “I just keep learning on the job. It keeps it interesting.” And while...topic, but I haven’t met any.” A few other tips: “Learn HTML, write a lot to make sure you don’t hate it...

...professional Addgene is a thought leader in scientific sharing, leading by example to increase sharing in...Resources on Addgene.org Learn More About Our Mission Meet the Addgene Leadership Team Get the Latest News...arts organization. Many people are surprised to learn that Addgene is officially filed, recognized and...is a nonprofit? The term "nonprofit" is a bit misleading. Nonprofits can generate revenue (as Addgene ... Xuan Additional Resources on the Addgene Blog Learn about Our Collaboration with Fellow Nonprofit Seeding...

...s antibodies are plasmid-based and recombinant. Learn more about recombinant antibodies here. Finally,...Finally, Addgene is a nonprofit that connects colleagues and is invested in improving research infrastructure... to maximize the reach and impact of your work. Please email [email protected] if you would like to ...plasmids via Addgene! Email [email protected] to learn more or get started.  If you have suggestions about...ideas for new materials to add to the collection, please let us know by filling out this suggestion form... will find useful antibodies for your research. Please consider sharing your experiences with antibodies...Resources We would also like to acknowledge our colleagues at the Developmental Studies Hybridoma Bank, ...

...Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts in your experiments. Read this ...Professor at Albert Einstein College of Medicine, to learn how problematic oligomerization can be and how to...that occur during development. Read this post to learn more about recombinase-based state machines and ... Green (both formerly at the Wyss Institute) to learn how these amazing tools may be used to rapidly diagnose...you can use to study basic virus biology while leaving virulent components behind. Minigenomes are stripped...and transcription as well as reporter genes. To learn how you can use minigenomes in your research, check...