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...webinars, and learn more about the field.  Read the CCST Science Policy Career Guide to learn more about ...field from a more traditional policy background, learning the science part on the job. Each path to science...policy professional over Twitter or LinkedIn to learn more about their career trajectory. If there’s one...one role can encompass many of these activities leading to a rich day-to-day work life.  Action Items:  ... profession. Figure 1: Science policy can lead you to many paths! For a more details, check out...The big secret about science policy that I didn’t learn until arriving here in DC, is that so much policy...little P” policy, the possibilities are endless leading to many ways for you to find your own niche.  Action...

...These very cute birds learn their songs like humans learn speech. Finches must learn early, use auditory...branches of the evolutionary tree at least 6-7 times and on at least 2 separate continents. Mimulus (Andrea...and I met scientists from 21 different labs who clearly also share this wonder of the organism.   It’s...beyond obtaining “omic” data and had to include clear plans to connect Genotype to Phenotype thus making...) -  perhaps you’ll choose to study Crepidula atrasolea or Prochlorococcus with your next project. Gymnotiformes... and if she decides to raise their offspring or leave them to their own devices. Chytrid fungi (Lillian... aspects of cognition. Spiralians - Crepidula atrasolea (Jonathan Henry) This black footed slipper snail...

...field, requiring people who are willing to quickly learn, and apply their knowledge to a variety of problems...problems. In this post, I’ll share what I’ve learned as a healthcare consultant and give you an idea of what...activities depending on the expertise of the senior leadership and members of their consulting staff. Who would...The consultants who enjoy their jobs most, get pleasure from taking up such complex challenges, quickly...ameliorated by thoughtful planning and engagement, leaving mental energy to enjoy the more rewarding and educational...in Bioengineering, and one of the things that pleasantly surprised me in my first few months as a healthcare...molecular behaviors of chemicals and proteins to learn how a particular drug or medical device works. This...

...been used since - including: mCerulean/mVenus, mCerulean/Amber, mCerulean/SYFP2A, mTurquoise/mVenus and... fused to the N-terminus of monomeric Cerulean mCerulean C1 Cyan Mammalian Express a gene of interest...microscopy (FLIM) method. Acceptor photobleaching method The acceptor photobleaching method is simple but limited... quenched when FRET occurs. By photobleaching the acceptor, you release the donor's quenching and the ...also been used to measure conformational changes, cleavage activity using FRET-based biosensors, and interactions...Mammalian Expresses mammalian optimized Aquamarine mCerulean N1 Cyan Mammalian Express a gene of interest fused...interest fused to the C-terminus of monomeric Cerulean mTurquoise2 Cyan Mammalian Constructs to target mTurquoise2...

...specific DNA sequences and companion endonucleases that recognizes and cleave the same DNA sequence if not methylated... questions is the same--methylation! Read on to learn about how DNA methylation may affect your restriction...restriction digests. Why methylate? Restriction enoducleases are so ubiquitous in the lab that it is easy...prevent them from being degraded by restriction endonucleases. A given prokaryote typically has genes encoding...whether certain restriction enzymes can effectively cleave DNA. Common lab E. coli K12 strains such as DH5alpha... activating enzymes such as DpnI. For example, cleavage by XbaI may be blocked due to methylation if the...results that would either be difficult to interpret or lead you to conclude your plasmid is not correct! Conversely...

...mentees efficiently Allows all group members to lead and learn at the same time Broadens the network of the...).  Assign each meeting a leader from the group in a rotation. The leader for that meeting is responsible...choosing a topic and leading the discussion. This is a chance to practice leadership and communication skills...Harvard Medical School researcher Connie Cepko to learn about her mentoring style. While working on this...intrinsic to their success. Their stories made it clear to me that, executed correctly, the group mentoring...a similar development stage, they have a lot to learn from each other. Some have taken my advice and I...forming your own peer mentoring group Find 5-8 colleagues that you think might be interested and willing...

... of CRISPR/Cas9 by altering the nuclease activity of the Cas9 nuclease itself. Design and testing of eSpCas9...improved the on-target specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1,... eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), Slaymaker et al. hypothesized...charged groove formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand... separation and target DNA binding to the Cas9 nuclease. Stable strand separation is maintained through...genomic sites. Knowledge of Cas9’s dynamic structure leads to the development of HypaCas9 Researchers in the... which portions of Cas9 might be important for nuclease specificity. They found that eSpCas9(1.1) and ...

...the learning of many facts but the training of the mind to think something that cannot be learned from...research, they gain confidence and learn to work independently, which can lead to greater success in meeting...take a different approach. His own most impactful learning experiences happened in the lab, and he wanted...settled on a two-semester model, in which students learn to express, purify, and analyze MDH in semester ... work and become more actively involved in the learning process. As trainees become more comfortable with...trading structure and predictability for improved learning outcomes and the opportunity to make new discoveries...department. So in 2016, Provost partnered with colleagues at other institutions to form a support network...

...you can learn more about possible strengths and weaknesses, and view everything as a learning experience...project she needs one of you to lead. At first you might think, “I’m not a leader,” because you don’t see yourself...develop your nascent leadership skills. You might just find that you’re more of a leader than you thought!... portion of the interview out of the park. It’s clear that you know how to do the job. You may think this...Stop! Take a step back and think of a time you’ve lead your team in a defined direction in the past. Maybe...opportunity much different? Might you already have leadership skills that are just not being tapped at the ...time to write down all the skills that you have learned and that you frequently have to exercise in order...

...college, and science, are places for learning and growth! As you learn more about yourself and the world ...This post was contributed by guest blogger Eleanor Wang, a Research Specialist in Patrick Hsu's lab at...science, experimental design, and execution. Can lead to an independent project. Lab technician / ...Talk to your professors about their research, and learn about what makes them excited to do what they do...what you’re working on is important! Better than leaving yourself in the dark, but if you never ask, you...also more than eager to help you along your path. Learning about their career paths and goals helped me reflect...concept to me when I was in high school, but I’ve learned the value in finding mentors wherever I go. This...

...gaga over the newest gene-editing protein - a DNA-cleaving Argonaute from Natronobacterium gregoryi, or NgAgo... CRISPR proteins Cas9 or Cpf1 - keep reading to learn more! Background: Argonaute and NgAgo In eukaryotes...role in RNA interference, binding RNA guides to cleave foreign RNA. Argonaute proteins are also present...thermophilus (TtAgo), showing that this protein could cleave DNA in vitro, directed by phosphorylated DNA guides...Swarts et al. were unable to show TtAgo-mediated cleavage in mammalian cells, likely due to high temperature...Argonaute. They identified NgAgo and showed that it can cleave plasmid and genomic DNA in a variety of mammalian... 10 genes, they did not observe differences in cleavage efficiency. If this finding is replicated in larger...

... 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...

...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...

...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...

...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...