...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...

...students, joining a new lab at any career stage, learning a new technique while visiting a lab, or maybe...trainee practices gives twofold benefits: you may learn faster and retain more information plus your mentor... your experience in the research group more (at least at first) than any other one. Luckily, you can do...familiarize you with the techniques you will be learning and the biology you will study. (You can also ...details, especially when you are in a new place, learning something new. So how can you remember everything...the procedure. Be an active partner Everyone’s learning style is different, so let your mentor know what... review their notes, process what they’ve just learned, and ask questions later – maybe even the next ...

...Although CRISPR-Cas12a, with abilities to process and release its own gRNAs from a single array, has been a top...prime editing, both and their derived tools are leading the edge of the precision gene editing. CRISPR-...and has previously been used for multiplex Cas9-nuclease editing. Typically, a single array encoding the...array. After expression, the individual gRNAs are released by endogenous tRNA processing machineries, specifically...tRNAs Luckily, a fun feature of tRNA was fully unleashed by the Gao Lab’s work. Led by PhD student Qichen...promoter, which is followed by tRNA processing to release individual gRNAs for gene editing. They named this...human or plant source), by tuning the length of 5’ leader sequences of a given tRNA to achieve the highest...

...the manager to succeed. More people in leadership roles leads to more ideas, initiative and creative ... Are the desired goals and outcomes clear? If you don’t have clear outcomes in mind, you won’t be able... Series Learn How Volunteering at a Professional Organization Can Help You Develop Leadership Skils Set...Harvard Medical School researcher Connie Cepko to learn about her management style. Recipes for delegation...assignee and this is a recipe for bad outcomes. Clearly articulate the desired outcomes. Begin with the...delegate so the person doing the task will have to learn and stretch a little, but be careful not to go too...calendar even if it is just 15 minutes a week. Clearly identify constraints and boundaries. Where are ...

... the principles of gating, you’ll first need to learn a little bit of theoretical background on the different...might have to do some testing or ask experienced colleagues.   Figure 1: The laser pulse illuminates...prior to running my flow analysis, I can however clearly identify my lymphocyte population (10.7% of all...to generate signals in a reasonable range. If colleagues in your group already have experience with using...my negative, unstained population, which I can clearly differentiate from the positive one (Figure 8).... that in many cases a stain will not result in clearly distinct positive and negative populations. Oftentimes... majority of my cells of interest lies within a clear negative population. When you’re interested in measuring...

...First, it uses a dedicated 5’ exonuclease instead of using the exonuclease feature of T4 DNA polymerase...restriction sites (Type IIS restriction endonucleases) to cleave DNA outside of the recognition sequence..., Jin S, Reiser M, Lockey RF. CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless... was first described by Dr. Daniel Gibson and colleagues at the J. Craig Venter Institute in 2009. Here... which contains three different enzymes: an exonuclease, which chews back the 5’ ends of the fragment...over 200 nucleotides. This may be because the exonuclease could chew through an entire fragment shorter...Cas9 enzyme along with a specific gRNA was used to cleave the 22kb vector. When followed by the standard ...

...lookout for friends or colleagues that have the training or skills you want to learn. Mentors can be senior... “Learning is not attained by chance, it must be sought for with ardor and attended to with diligence...once you have found someone you admire and want to learn from. Check out Joanne's Reddit AMA Who should ...or coffee with someone I don’t know very well at least twice a week. I have done this with people I work...Harvard Medical School researcher Connie Cepko to learn about her mentoring style. Mentors at home and away...or see your work product in action) Can help you learn the local “ropes” and become an insider faster ...this Catalyst Report “Making Mentoring Work” to learn about what makes a good mentoring program. If your...

... had always been a student and I truly enjoyed learning.” Addgene’s Director of Product Management Eric...thing I wanted more of [after undergrad] was to learn and speak the language of science. I didn’t come... me, “it made more sense to get my master’s and leave. I was really devastated when that happened. I didn't...Instagram profiles here (Kenkel, Rinaldi)! But leaving a PhD program doesn’t mean you’ve failed. “I got... see where my life will take me. I’m also just learning. I have a huge interest in data science,” which...the lab, she told me, “I was really interested to learn more when I was introduced to bioinformatics in ...open and collaborative.” She knew she wanted to leave academia, and once she joined an investment club...

...back in 2007, which function as centres for peer learning and knowledge sharing, in the form of workshops...maintains a list of active spaces near you.   Learning about C. elegans with the OpenWorm Project One...variety of aspects from the molecular biology to learning how to grow and care for the worms. As an example...relies upon biologists and computer scientists learning and communicating with one another.      Currently...optimizing the health of domesticated cats and learning about feline microbiomes. One of the aspects of...the kittybiome project, but the primary goal is learning in this case.   Funding Makerspaces One of the...controller and made it spin again!  We’ve also learned to be resourceful and to repurpose old equipment...

...capable of cleavage, they tested the clones against an NNN PAM library in bacteria where cleavage leads to the...Addgene Blog Learn about Engineered Cas9 PAM Variants Read about CRISPR Base Editing Learn about Homology...increased editing specificity. We’re excited to learn more about xCas9 - here’s what we know so far! ...GAT, GAA, and CAA PAMs, they saw that xCas9 3.7 cleaves multiple PAMs at a higher efficiency than SpCas9...xCas9 are similarly flexible. Like the xCas9 3.7 nuclease, xCas9 3.7 fused to third generation cytidine ...modifications that can be made to Cas9. It is a pleasant surprise that selecting for one beneficial trait...Guide Find CRISPR Plasmids for Mammalian Systems Learn about CRISPR History ...

...strengthen relationships with your colleagues and supervisors and lead to you being remembered as a person... fill this gap by highlighting that researchers learn many transferable skills while working in the lab...most straightforward, it can mean that everyone leaves disappointed (we’ve all won an argument but felt...sometimes there’s no agreement at all and everyone leaves unhappy.   Negotiation as a creative problem-solving...underlying reasons for this person’s request? Your colleague is if someone can feed their cells this weekend...maintaining and growing relationships with your colleagues within and outside of your organization. In my...still being able to take my vacation? I want to learn more about a different area of the company. How ...

...working in the laminar airflow are also among the leading causes of contamination. These aerosols generated...naked eyes and hence cannot be detected until it leads to contamination. Another source of the problem ...the cells should be in continuous culture for at least two weeks to give time for low levels of contaminations... the medium should not have been changed for at least two or three days before sampling (Uphoff and Drexler...result interpretation. Also, the signals from the nuclear area make the detection of mycoplasmas difficult...realize it. Here are some things to do: Wear a clean lab coat and mask while handling cell lines. Avoid... cross-contamination. CO2 incubators should be cleaned regularly and dampness of any kind should be avoided...

...reversibility of the genome editing process. Read on to learn about the various light-controlled CRISPR tools ... Sato lab unveiled a photoactivatable system to cleave a target DNA sequence (Nihongaki, et al., 2015)...system is unique in that it utilizes a “split” nuclease - the authors fragmented Cas9 into N-terminal ... moieties move apart, allowing DNA binding and cleavage. This technique was applied to create photoswitchable...inhibited by the unfolding of LOV, allowing the release of Cas9. A noteworthy application of the CASANOVA...in U2OS cells. Upon light activation, dCas9 is released within minutes and red foci formed 20-40 min later...PCK upon reaching the amber stop codon, TAG. (To learn more about site-specific incorporation of amino ...

...and concentration of released DA in mouse brain slices Visualization of DA release dynamics during locomotion... hormone,” it’s also involved in reinforcement learning, decision making, and motor control.  What do...Though DA firing patterns (or the frequency of its release) are well understood, the direct effect of DA binding..., enabling detection of individual peaks of DA release.  Using dLight1 sensors in experiments The Tian... mice Report modulation of DA signaling during learning and reward consumption in live mice Find dLight...following types of data: Report endogenous DA release from a single dopaminergic fiber in mouse brain...stimulus in vivo in transgenic zebrafish Report DA release in live mice over the course of Pavlovian conditioning...

...Your task is to convince them to accept you as a colleague and give you a million dollar startup package....experiments or a laundry list of molecules to screen has unclear utility. You cannot predict what will happen. Without...validity of a single model. The first approach can lead to new directions, while the second approach could... terminate with the rejection of a model and no clear alternatives. Typically, a research vision is broken...knowledge gap. Aims should have short titles, a clear easy-to-understand question/goal, an approach, anticipated...looks like. If you are successful, what will be learned and/or now be possible to do? Research accomplishments..., anticipated outcome, and payoff for each aim. Learn to communicate your story with only the most salient...

...works with Addgene in some way. For users, we just released a major redesign of how search works on our site...and professional backgrounds. Sometimes this can lead to wildly different ideas of how to approach a problem...information. It is quite possible that the clarifications lead to the story being scrapped or broken up into smaller...After the code is written, it is reviewed by at least one other developer. This is another point of collaboration...different parts of the code. I think the best way to learn about how any code works is to poke it and see what...places, we might be calling it an aliquot. This can lead to confusion as to what the code is supposed to ...on a common name. While we do our best to write clean, reusable code when working on a feature, sometimes...

...genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015)... control for these faults. However, it’s still unclear if similar pitfalls exist for CRISPR screens and...off-target cutting of only a handful of guides. It’s unclear if off-target cuts confound the results of high-throughput...without a reduction of on-target activity, but it’s unclear if this holds true in genome-wide screens.   ... are used as controls. Less significant p-values lead to more false-negatives results (i.e. failing to...to identify a gene that’s a hit); but they also lead to fewer false-positives results (i.e. mistakenly...non-targeting guides as controls. While it’s not clear how safe-targeting guides will affect phenotype ...