... is a strict requirement for Cas9-mediated DNA cleavage. The need for more PAM sequences While PAM sequences...identify mutants of S. pyogenes Cas9 that were able to cleave target DNA sequences located upstream of either... to wild-type SpCas9, which also displays some cleavage with an NGA PAM. This variant may increase the...VQR, EQR, and VRER Cas9 variants are capable of cleaving genomic DNA in mammalian cells and zebrafish embryos...using wild-type SpCas9. The number of off-target cleavage events for the VQR and VRER variants is similar...genome engineering was Cas12a (formerly Cpf1), a nuclease that generates double strand breaks in target ...formerly C2c2) has been adapted for targeted RNA cleavage in mammalian cells. The REPAIR (RNA Editing for...

...further improved, by adding a nuclear localization sequence (SV40 Large T nuclear localization sequence) and...step, the two compatible target sites are cleaved out, leaving behind a vector with the inverted DNA flanked...Additional Resources on the Addgene Blog Learn More about FLEX-vectors Learn More about Cre-Lox Check out Retrograde... Cre expression have been developed. Read on to learn more about FLP-FRT, Cre-lox, and how combinations...asymmetric core. Recombination between these sites can lead to excision, inversion, and translocation of DNA...Continuous inversion of the DNA sequence of interest can lead to poor expression of genes within that sequence...between the two target sites with identical sequence leads to inversion of the flanked DNA sequence and the...

...example, learn about how researchers are using “big data” to look at Lyme Disease in new ways. Or learn about...otherwise never learn from the valuable research we are doing. Browse the site, learn from others, share... fails to reach our primary target audience – colleagues in our field. Secondary audiences that may also...therefore must discover new ways to reach our colleagues and other interested audiences quickly and concisely...articles as the dominant tool for sharing ideas. This leads to everyday obstacles for many of us, such as finding...can select the disciplines and the years. Which leads to the question of how we can support researchers... disciplines. But there remains a great deal to learn from the science of others. Improving science communication...

...Readiness Posts Learn Good Management for Scientists Resources on Addgene.org Learn about Careers at...Why We Support New Federal Overtime Rules, made clear that institutional arguments were not as convincing...rather than being centrally administered, which leads to many complications, including the inability of...Furthermore, 7% did not enforce these policies. It is unclear how many postdocs are at each of these institutions...they recommend NRSA levels, postdocs must be at least at the FLSA minimum of $23,660. Any postdocs on ...are afoot for the postdoc population, and it is unclear how this will all shake out. In the short term,...doing - for example, your benefits may be changing. Please also feel free to contact us if you have information...

...caspase-3 (an executioner caspase) cleavage site (DEVD). Upon cleavage of the sequence, β10 and β11 can ...protease cleavage site. TEV protease is a highly specific protease that is commonly used to cleave proteins...activate a cascade of caspases, protease enzymes that cleave proteins. Executioner caspases are activated last...degradation of over 600 cellular components ultimately leading to cell fragmentation and death (Elmore, 2007)....reporters, the FRET pair is linked by a caspase cleavage site. Cutting the linker by a caspase results ...changes of the donor and acceptor after proteolytic cleavage. Secondly, fluorescence imaging of living organisms...GFP fluorescence increased over time and showed a clear pattern in the developing forebrain and retina. ...

...slides, learn from audiences as they do science outreach and engage with the public, and learn from the...to practice communicating.  Excellent scientists learn from advisers and other mentors by collaborating... aspects of the work you presented may also be unclear. 2. Make the audience your primary focus When writing... to it - what will it make them think? Does this lead to the conclusions I want them to draw and the perspective...scientist needs to be able to communicate with their colleagues and peers. Scientists also need to be able to...in other fields, like when collaborating with colleagues in other fields or writing grant proposals to...to listen to you practice.  Ask them what they learned from your presentation and evaluate whether or ...

...surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda) 5...researchers instead turn to so-called “mismatch nuclease” assays (e.g. Surveyor® or T7E1) for mutation ...Digital PCR has several advantages over mismatch nuclease assays that will be elaborated below. Digital...advantages over the commonly employed mismatch nuclease assays. Sensitivity: Less than 20 ng of total...mutant targets have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between...functional knockout of your target gene. Mismatch nuclease assays are completely “blind” to this very important...gene-editing frequencies (GEF-dPCR) mediated by designer nucleases. Nat Protoc 11, 598–615 (2016). PubMed PMID: 26914317...

...for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage. Summary...methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked...), 3’->5’ exonuclease activity (increases amplification fidelity), and no 5’->3’ exonuclease activity ...repair, the template should be resistant to Cas9 cleavage. If a plasmid contains the template, site-directed...template is removed by DpnI restriction digestion leaving behind only the mutagenized plasmid. Although it...by the dam13(-) mutation – you’ll want to steer clear of these strains when preparing the plasmid template... cannot be used for site directed mutagenesis. Nuclease To remove the template DNA (unmodified plasmid...

...precision with dual-nuclease Cas9-Cas9 chimeras Scot Wolfe’s lab has developed dual-nuclease Cas9-Cas9 chimeras...Cas14 targets and cleaves ssDNA which they have adapted for detecting SNPs. Learn more from this Addgene...editing in plants While there have been many Cas nucleases developed for plants, it has been difficult to...to compare the performance of different nucleases. Nicola Patron’s lab compared the efficiency and specificity...multiple wild-type and engineered variants of Cas nucleases. In doing so, they also created a modular toolkit...light-mediated genome editing is now possible. Learn more from this Addgene blog post Read the paper...factor localization and binding via ChiP-seq. Learn more about ENCODE Find the transcription factor...

...you are also learning to be an excellent scientist. Leadership and management Take a lead from the amazing...completed a postdoctoral position… or two. You have learned a lot. Whether you are pursuing an academic career...transferable” skills you have developed in addition to learning how to be a scientist. Why not stack the deck ...science faster. Help other people when you can. The colleagues you help will be future advocates and references...in your department, create them and show your leadership skills too. Teach classes Go to workshops and...Technical knowledge and creativity Become a “thought leader,” develop a technical “niche,” develop a useful...Follow industry publications like FierceBiotech to “learn the business” Attend events with local networking...

...make it clear that you will be doing your research in a group or facility that is a world leader in your...area are looking for. Remember though, you need to leave them time to read it, give it back to you, and for...reading through your proposal, ask yourself: is it clear how novel my findings will be and why these findings...around to different labs during your career; you learn new techniques and interact with new people. If ...working for the last few years, you need to be very clear that you are staying because it is the best place...paragraph and point out words or sentences that are unclear. We’re so used to conversing with other scientists... panel. It’s important for this person to get a clear and concise explanation as to why they should give...

...that gives our customers the most utility in the least amount of time. Though I now spend far less time...buried beneath someone's lunch. Many times, I’m pleasantly surprised to discover that the most crucial information...website are another great source of information. At least at Addgene, the questions in our FAQ section are...from you and it’s wrong. Can you help us fix it? Please address as soon as possible!” and I always begin...sure to mention the plasmid catalog number, or at least the plasmid name in your initial query. This can...your concern is based on data you have produced, please share the data. Maybe not all, but many bio companies...helping us understand the nature of your concern. Please be courteous. We understand how exasperating it...

...successful experiments: To clean air and beyond Turn on the hood for at least 15 minutes before you start...start any tissue culture work and make sure that clean air is flowing in. The vent for the in-flowing air...things in and out of the hood while work is in progress.Learn more about cell culture contamination in this...this post from the ATCC Declutter! Keep the hood clean Hoods should not be used as storage cabinets. Clutter...Clutter not only makes it difficult to clean your workspace properly, but can also disrupt the laminar...(especially if you have air conditioning on) so leave them in a sterile incubator. This also means that...have a water bath constantly at 37°C for this purpose.Learn about the effects of CO2 on sample pH Cell morphology...

...confused because the HNH nuclease active site wasn’t even close to its reported cleavage site on the complementary...and shows the HNH nuclease domain swung around in close juxtaposition to its cleavage site on the target...students work closely with local researchers to learn about their research while creating physical models...protein in a conformation that was not “poised for cleavage”. Jacob computationally added the non-template...model is certainly useful, it is also wrong in at least one serious way. We represented the Cas9 protein... DNA strand, and then activate two different endonuclease active sites. Fortunately, there is never just...on Fuguo Jiang’s structure of Cas9 – primed for cleavage. This structure captures the displaced non-target...

...thermostable periplasmic endonuclease that can sometimes co-purify with your plasmid DNA and lead to degradation...Visit our Plasmids 101 Topic Page Learn How to Verify Your Plasmid Learn about Common Lab E. coli Strains... on How to Quantify Your DNA Learn to Do a Bacterial Transformation Learn How to Perform a Diagnostic ...recommend performing an extra wash step to remove endonucleases (see figure at right for an example). Look out...Certain strains, including HB101 and derivatives, release large amounts of carbohydrates that can inhibit...adding supplements can improve your cell density, leading to an improved plasmid DNA yield. Culture conditions...directly from a frozen glycerol stock or agar stab may lead to loss of the plasmid, and using older plates could...

...Resources I asked some colleagues to share valuable life lessons they have learned during their scientific...out what you want to do after graduate school, at least not while you’re still in the thick of it. About... start CINSO. Here, I share some of the tips I learned along the way and hope they’ll help you make the...made a mistake, I evaluated what lessons I had learned – call it “troubleshooting.” Trying out new experiments... example to use to promote future attempts and learned a bit about recruiting scientists to outreach.  ... of these resources as I could. Networking and learning from the careers of others were key to creating...share science with the public, but I can watch and learn from the experts in science communication in real...

...to insert an endonuclease gene within its own cut site1. In heterozygotes, the endonuclease cuts the wild-type...accidental gene drive release, which would represent proof positive that at least some scientists could...ensuring that all future offspring inherit the endonuclease gene. As we pointed out last year2, delivering...problems in health, agriculture, and conservation. But please be aware that there are many easy-to-implement ...building candidate gene drives intended for eventual release should consider building an appropriate immunizing...mitigate the potential effects of an accidental release3. Which gene drive confinement strategies should...Against this, consider the cost of an accidental release. Science relies on popular support, which in turn...

...droplet in each well (Figure 2). A clean ddPCR should have a clear separation between positive (blue) ...and tricks for performing ddPCR Clean everything You must have a clean working area to titer your AAV .... Here are some steps you can take to achieve a clean NTC: Have a dedicated bench with a dedicated set...Wipe down the bench and all consumables with 10% bleach.  Pipette slowly If you are using a manual droplet...This will reduce aerosols that could potentially lead to contamination. Optimize your PCR For a good starting..., 2014). If you are having difficulty getting a clean separation between positive and negative droplets...amplification, your fluorescence amplitude will increase, leading to greater separation of your positive and negative...

...CUT&RUN, which stands for ‘Cleavage Under Targets and Release Using Nuclease,’ was recently developed by...for its nuclease activity and is chelated from the reaction up until this point. The nuclease reaction...Additional Resources Learn how you can use FRET to study protein interactions Learn about other types of...cutting activity of a Protein A fused micrococcal nuclease (MNase) to specifically isolate DNA that is bound... MNase has been localized to target sites, the nuclease is briefly activated to digest the DNA around ...digestion. After MNase digestion, fragments are released from nuclei by a short incubation at 37 °C. These...

...willing to learn and grow in skills. Managing a team is hard and most people have some learning they need...still not get the most from your team. It is pretty clear that if people are mostly happy at work, they will...think it, you have to tell them. A good manager learns that her success and recognition come from the ...and diversity built into their jobs Chance to do/learn new things, diversity of responsibilities, which...their job. Good relationships with clients and colleagues Sometimes this means you have to fire a good ...scientists) Are self-directed, but need precise leadership and support from their superiors (beware of not...scientist if you want their best work) Are continuous learners and have individual priorities for advancement...