...reproducibility at Addgene and here I summarize what I learned both in preparation for my talk and at the minisymposium...science and research. Nonetheless, I’d like to steer clear of saying we have a reproducibility “crisis” on ...irreproducibility with some solutions based on what I’ve learned. I hope you can apply some of these solutions to...interactive protocols that make it much easier to learn new experimental techniques. Electronic lab notebooks...perfect story for a “high impact” publication can lead to the publication of rare flukes showing positive...begin requiring reproducibility checklists that at least make it obvious that reproducibility is a priority...performing experiments with too few replicates leading to suspect statistical significance (you can read...

... in order to protect their DNA from host cell nucleases (4). To further complicate matters, even when ..., and Szymanski in January 2016 in Viruses (5). Please refer to the paper for more details on the assay...short recognition sequence (like MluCI) that will cleave DNA frequently enough to result in a smear of fragments...inserts. We consider a library worth using if at least 40% of colonies tested showed correctly sized inserts...membranes will then be exposed to a lysis buffer to release recombinant proteins directly onto the nitrocellulose...solution-soaked filter paper (colony-side up) and leave for 1 h at room temperature. After this, it is helpful...min at 37°C (each repeated 5 times) works well. Leaving the membrane overnight in the freezer during one...

...paste in the genome. The Cas9 nuclease has since been altered to a nuclease-dead Cas9 (dCas9) by introducing...introducing mutations in its nuclease domains. dCas9 no longer cleaves DNA but still binds to specified genomic...Albert Cheng and Mark Wanner. CRISPR-Cas9 offers a leap forward for genome editing, providing researchers...expression vectors available through addgene.org); at least one sgRNA-PBS (single-guide RNAs appended to one...experiment. For updates and more details about Casilio, please visit http://casil.io and the Addgene Casilio page...101 Posts Read about Other Applications of CRISPR Learn How to Design Your gRNA Additional Resources on...

... not be too leaky. For inducible prokaryotic promoters, pLac is known to be slightly leaky, and pBad is..., and light are all examples of factors that can lead to the induction of a promoter. Below, you’ll find... cAMP and represses pBad, decreasing promoter leakiness.   Other examples of chemically induced promoters...sensitive expression systems are typically less leaky than chemically induced promoters; they show near-zero...heat shock. In the case of Hsp70, the heat shock releases heat shock factor 1 (HSF-1), which subsequently... be compatible with your experimental system. Leakiness If you’re expressing a gene that may be toxic,...inducible promoters are also known for their low leakiness. Inducibility level How much do you want to induce...

...Cell Stem Cell, Musunuru, Chad Cowan and their colleagues describe a much more efficient tool for doing...cells: a transcription activator-like effector nuclease (TALEN) kit consisting of 834 plasmids. The researchers...proteins that comprise a DNA binding domain and an endonuclease domain. What’s distinctive is that the DNA-binding... rest of the protein in place including FokI endonuclease domain as well as fluorescent proteins used ...first really popular tool was the zinc finger nucleases. Unfortunately, it is time consuming and difficult...without a lot of expertise to create zinc finger nucleases that will bind desired sequences. TALENs are much... genome. It remains to be seen which system is cleaner in terms of minimizing off-target effects, but ...

...Resources on the Addgene Blog Learn More about Homology Directed Repair Learn More about Non-Homologous End...editing team at the Allen Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org...based on known functional aspects of the protein gleaned from the literature and engagement with researchers... Institute for Cell Science Plasmid Collection, please visit the Cell Catalog on the Allen Cell Explorer...hiPS cell lines generated using these plasmids, please visit the Cell Catalog on the Allen Cell Explorer... study cellular structures and processes and can lead to the better understanding of cell biology. We ...

...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. ...

...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   ...

...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...

...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...

...Recently I learned that Addgene’s pLKO.1 cloning protocol is viewed around 3,000 times each month. I ...this plasmid, and if it still being used. After learning of it's continued popularity, I decided this protocol...thousands of other scientists using pLKO.1 variants learn about those updates? This is a problem many scientists... found in our Plasmids 101 blog series. Want to learn more about sharing your plasmids via Addgene? Check...lab for non-bench work – but is there a sterile, clean way to bring your phone into the lab? Maybe having...protocols to the site and will keep you updated as I learn to use the resource. OpenWetWare Wikis Website: ... using. For the novice, the format does have a learning curve, but OWW walks you through the process with...

...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 ...

...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...

...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...

...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 ...

...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 ...