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...bluntly with WT Cas9 and one gRNA, you can create a staggered cut using a Cas9 nickase and two gRNAs.  For ...test nickase preferences using HEK293 cells. They started by comparing D10A and H840A total editing efficiency...PAM-in and PAM-out configurations with varied nick distances (40 - 130 nt). From these results, it’s clear ...than that of H840A, especially with smaller nick distances. Using D10A, they subsequently showed that editing...low when the gRNAs are too close (7-23 nt nick distance). Both D10A and H840A are potent editors, but ...obtain high HDR efficiency. Nickases create a staggered cut - could this system mediate repair throughout...the target site rather than the optimal <10 nt distance. Employing two gRNA sites that are further away...

...alternative loading control option. Total protein stains or stain-free imaging systems are both effective for...440, Gilda, J. E., & Gomes, A. V., “Stain-Free total protein staining is a superior loading control to β-actin...Gilda, J. E., & Gomes, A. V. (2013). Stain-Free total protein staining is a superior loading control to β-actin....” Fully quantifying a western blot requires a standard curve with a pure sample of your protein of interest...validate it for your particular setup. We recommend starting by searching the literature to see what is already... Weiler, I. J. (2008). The use of total protein stains as loading controls: An alternative to high-abundance...

... are a few out-of-the-box ideas that might jump start your learning. 1. Create a peer mentoring group ... career and life goals. For tips on how to get started see Form Your Own Peer Mentoring Group: A How-To...for the live audiences in your future.     3. Start something In almost every career talk I present,...charge before others do”. Candidates who have “started something” which they can share on their resumes... of things: led a grad or postdoc association, started a speaker series or biotech club, managed their...ukelele...perhaps you get the idea. What will you start? 4. Do informational interviews (on a schedule) ...you can’t wait until you imminently need a job. Start now. Informational interviews are 1:1 discussions...

...named NeuroStack was used (Orguc et al., 2020). Using a programmable microcontroller, NeuroStack coordinates...admittedly for self-serving reasons – I want to understand my obsession with the combination of chocolate...connection to study on a molecular level. The brain is constantly influenced by signals it receives from the entire...digestion of food, while brain tissue is relatively static). To operate both types of implanted fibers simultaneously...stimulation in target tissues of a live animal. They started by delivering an AAV construct expressing channelrhodopsin...tissue. They performed optical stimulation using NeuroStack and the fibers as previously described, then ...implantation of fibers, indicating the system was indeed stable for continuous monitoring. On the gut side of things...

... MIT's Ed Boyden explains it, his goal is "to understand the brain at a level of abstraction that enables...Once scientists can do that, they will not only understand how the brain works, but also how to fix it when...then alongside blue they actually have a lot of crosstalk. So we developed a red molecule. We also had to... use dim blue light and red light together. Understanding the brain through optogenetics research Addgene...areas of benefit: First, there is fundamental understanding of the brain, which has humanistic and philosophical...region, the animals became aggressive. If we can understand the neural basis of violence or aggression, it... neuroscience has so many different goals: to understand how a mouse changes emotions, to see how a monkey...

...This system ablates tumors just as effectively as standard CAR-T cells, but it reduces the off-target effects...expression (or the eGFP proxy for CAR) is elevated at stable levels for 6 days - after just 15 minutes of FUS...PMCID: PMC6377228. Hyperfolder YFP- a chemically stable tool for microscopy by: Susanna Stroik Microscopy...these applications have lagged. Fixation, chemical staining, and light exposure can negatively affect FPs ...hfYFP is not only brilliantly bright, but it also withstands denaturing conditions, high temperatures, and...fluorescence resilience to these preparations. After crystalizing the hfYFP structure, the group rationally designed...similar capabilities. If you’re looking for a super stable FP for your next microscopy experiment, consider...

...easily started and easily credited. And nothing stops these one-time favors from becoming starting points... rewarding way of making science.   How we started Crowdfight-COVID19 Crowdfight was born as a collaborative... all over the world had signed up, and soon we started receiving dozens of requests every day.    ...suitability and availability; 3) Make the contact to start a direct collaboration between researcher and expert...are undergraduates or PhD students who are just starting their journey into science. Others are experienced...Crowdfight is also open to other topics.  In this new stage of the project we are focusing on what we call “...-and-error. A recommendation for the correct statistical test can save a retraction. Today, collaborations...

...exquisitely important in the brain. To better understand how astrocytes and neurons interact, the labs...their powers for better or for worse. To better understand this transfer, the Simon Grelet Lab developed...that this transfer increases the metabolic and metastatic capacity of cancer cells. This is certainly just...Nerve-to-cancer transfer of mitochondria during cancer metastasis. Nature, 644 252–262. https://doi.org/10.1038...had better brightness, signal-to-noise ratio, photostability, and signal response upon Ca2+ binding. Most...soma-targeted variant SomaFRCaMPi also performed substantially better than other soma-localized red GECIs, ...calcium sensor with exceptional brightness, photostability, and multiplexing capabilities. bioRxiv 2024.12.16.628673...

...lab members stay around? Is it too long or do they leave prematurely? Do lab alumni stay in the same ...why not? It may not be a bad thing if the lab has stayed that size on purpose, but it could also be a sign...is workplace bullying? Threats to professional status – including belittling opinions, public professional...discipline or competence procedures Threats to personal standing – including undermining personal integrity, destructive...impossible deadlines, unnecessary disruptions Destabilization – including failure to acknowledge good work... observe grant writing? Get equipment quotes? Understand administrative responsibilities? Is the work ...on more than one project at a time or over their stay in the lab? Do all lab members attend scientific...

... beneath it. You can jump to the different time stamps in the description to watch any sections you’re...watching the whole livestream for a more holistic understand of reproducibility issues and their potential...limits of knowledge as they develop new ways of understanding how the body works, generating new therapeutics...have intricacies that aren’t always mentioned in static written protocols. It is therefore a bit ridiculous...reproducibility is making sure that others can understand your analyses and repeat them. Companies like...initial hypothesis through validation are easier to understand. The need for great stories has, however, made...experiments with too few replicates leading to suspect statistical significance (you can read more about this problem...

...causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability. Cell...Multimeric plasmids are also considered to be less stable and more prone to being lost in bacterial cells... of multimer present in your sample. In these instances, please note that the supercoiled dimer band can...1981). E. coli strains such as DH5α, HB101, NEB Stable, Stbl3, etc., are recA- mutant strains, and therefore...the transformed bacterial culture into the late stationary phase. Growing the culture at a lower temperature...R, Walther W, Michel ML, Schmeer M. Long-term stability study and topology analysis of plasmid DNA by ...direction are the secrets of multicopy plasmid stability. Mol Microbiol. 1998 Sep;29(5):1137-45. doi: 10.1046...

... our scientific support staff receives – in fact, the blog originally started as a way to easily store...Thus, we are constantly updating our content to reflect the newest breakthroughs and standards. It’s always... be surprised to learn that Addgene’s mission statement extends beyond plasmids, AAVs, and antibodies ...in the spring of 2022. The first step was to understand information our guide needed to have. Through...someone new to antibodies might need to successfully start using them at the bench, along with questions we...Making content is an iterative process. We are constantly reviewing the resources we currently offer to...

...repeating your work or making similar mistakes in the future. Stay organized Set up a table of contents...computer at the command line, and so was really starting from the beginning. Luckily, a fellow graduate...that it is helpful to those of you just getting started. The practices suggested here may seem tedious ... the analyses: Goal: Before you begin, briefly state the goal of the analysis, including background on... you to perform this analysis. Having a clearly stated, specific goal for each analysis will help you ...computer and is just another name for a folder. For instance, your Desktop directory contains the folders and...code does are very useful for those that are just starting out, and helps clarify the role of the code. ...

... microorganisms, yeast have both a stable haploid and diploid state which is useful for genetic analysis...will catch you up on the core replication and resistance features of yeast vectors and explain how they...the yeast chromosome; however, they tend to be unstable and may be lost during budding. Yeast Centromere...single copy. Unlike the ARS vectors, CEN vectors are stable without integration. Yeast Episomal plasmids (YEp...natural yeast plasmid) allows for 50+ copies to stably propagate per cell. The copy number of these vectors... due to high rates of spontaneously occurring resistant mutants and the insensitivity of yeast strains...auxotrophic markers. The MX series of antibiotic resistance cassettes is most commonly employed, with the...

...sample into the appropriate stab culture Grow the stab culture Ship the stab culture to the proper location... track the location, quality, growth, and legal status of thousands of plasmids a day, like we do at Addgene...some require going back two steps, some require starting over entirely, and our software needs to handle...corresponding entry in your database. Enjoy your happy lab staff. Barcoded materials make everyone's life easier... tricky procedure failed and one of your techs started over with a new tube. In the first example we’... valid technical reasons to choose a non-Python stack, but if you're working with scientists it's unlikely...

...other references to scientific literature to substantiate the facts upon which it builds. This means you...to relevant primary research articles. Then just start reading and following citations through the literature...Reading primary research literature The best way to understand scientific papers is to practice reading them...give you just enough information to allow you to understand the research question that will be addressed....Is the data of sufficient quality (error bars, statistics, clarity in images etc)? Did they perform all...publication, it doesn’t mean that there are no mistakes or misinterpretations. After all, the science ...research area. Set a goal for paper-reading, even starting out at 1 paper/week will be useful. Check out ...

...immunogenic proteins generated Stable integration into host genome and stable expression of the transgene... generations of lentiviral vectors have been established so far, with safety increasing in each generation...stomatitis virus G protein (VSV-G) that improves the stability and broadens the cellular tropism of the viral... In this case, your gene is repressed in steady state and is expressed when Dox is added to the culture...particles expressing proto-oncogenes or oncogenes for instance should be manipulated with care. See the NIH’s...well. Quickly after transfection (4-8h), cells start releasing viral particles into the culture supernatant...found very effective for T cells transduction for instance. I hope you will find this information useful...

...inbox. Best uses Staying up-to-date on new publications in your field. Feeds are constantly updated, so you...challenge is finding relevant papers. The latest installment of the Early Career Researcher Toolbox will highlight...articles related to your actual interests while also staying on top of the ever expanding body of biomedical...characters! Using Twitter to find papers  You’ll need to start following relevant accounts in order to turn Twitter...Scientists: Following other scientists helps you stay on top of what others are studying and gives you...keywords. It’s sort of like a keyword search that’s constantly updated. This Twitter list contains >300 Twitter... papers are published, similar to Facebook or Instagram. Key features Setting up a personalized feed is...

... embryonic stem cell stage using one of several techniques. The longest standing technique is the injection...Mice are a common model organism used to understand mammalian traits and genetically engineered mouse... to create engineered mouse models at embryonic stages, and the pros and cons of different genome editing...experiments. Inbred mice are particularly useful for understanding a specific effect related to an experimental...positive selection marker such as an antibiotic resistance gene is also included to select for successfully...which is then implanted into a female animal for gestation. Nucleases that induce double-stranded DNA breaks...which is then implanted into a female mouse for gestation. All three can introduce multiple base pair deletions...

...trials has been outstanding, with almost all patients experiencing sustained and substantial relief from ...clinical trial for SCD in 2022 and Editas Medicine starting a Cas12-based clinical trial for SCD in 2022 as...targeting the diseased allele specifically. Early-stage trials showed a high success rate in clearing misfolded...above! There are many, many more in the earlier stages of the pipeline, ones that have experienced hiccups...diseases, blindness, and diabetes. Directions and obstacles for future CRISPR therapies CRISPR technology ...systemic approaches, has its own unique set of obstacles. Thus far, several targeted therapies have taken...