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... D., Stamnes, S., Holt, K., Sinn, P., May, A. P., & Paulk, N. K. (2020). Methods Matter: Standard Production...neuroscience Let’s consider my first challenge when I started working with AAV vectors. I wanted to use an AAV...spread. (D-F)  High-magnification images immuno-stained against dopamine beta-hydroxylase (DBH, magenta...Addgene Data Hub is designed to help scientists understand how our vectors perform in various experimental... end of the year, those that meet our minimum standards (clear and detailed methods and image descriptions...available through the Data Hub. These minimum data standards are designed specifically to allow scientists ...Gao, G., Mueller, C., & Flotte, T. R. (2015). Stability and compatibility of recombinant adeno-associated...

...there are more! Engineering disease resistance through gene stacking, increasing production of a protein... uses of MoClo and the kits Addgene offers to kickstart these applications, no matter what your model ...isolation, then the MoClo Pichia Kit will get you started (Obst et al., 2017)! It is specifically curated...reporters (Engler et al., 2014). This kit is a great starter for your general plant cloning experiments. Addgene...basics’ MoClo plant kit buddy). It is important to understand your experimental needs before choosing one or...with a large library of common parts to get you started quickly (Fonseca et al., 2019). Recently, the toolkit...that backbone differences such as the order, or distance in the placement of plasmid elements can impact...

.... I started watching the questions and preparing answers early in the day. Although I didn’t start answering...people or discussing science, it is a great way to establish yourself as a thought leader and have real conversations...Software for Biology) on Reddit and got some substantial traffic that we thought it would be a good idea...definitely glad we did - you can see some of the stats from the AMA to the right - the Reddit community...description of our AMA and Joanne’s preferred time to start answering questions. r/Science has pretty strict...was posted at 8:00 am and Joanne didn’t have to start answering until 5 pm). The ability to see the questions...Fortunately, Reddit Science is heavily moderated. I started watching the questions and comments go up early...

...regulatory elements or use a non-canonical code (Gustafsson et al., 2004), such as Hoogsten base pairs and...could affect protein conformation, folding and stability, change post-translational modification sites ...actually be necessary for proper protein folding (Stadler and Fire, 2011). Therefore, codon optimization ...translation initiation (Dresios et al., 2006) and mRNA stability (Hausser et al., 2013) to name a few. This could... you may want to codon optimize before you get started. There are a ton of online resources available ...Biol 13:30–34 . https://doi.org/10.1038/nsmb1031 Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias...tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Research 28:292...

...inputs, such as the file name. We started small with a print statement and tested that the desired data...associated publications, but our quality control (QC) standards have evolved over the years to keep up with the...     Fig. 1: QC'ing data at Addgene is like standing under a Niagara Falls sized rainbow   Over...problems was deciding on the right system for us to start using. Prior experimenting with open source assembly...perform:      Fig. 2: Quick-stepping to a FASTA file   With our outline of individual steps ...Creating a bioinformatics workflow in Nextflow Establishing a complete pipeline however, required more than...libraries such as Pandas, Numpy, and Biopython.  We started at the usual recommended place: documentation. ...

...electrophoresis (PAGE) followed by silver staining or SYPRO Ruby staining and the molecular weight and relative...after negative staining. Empty vector particles can be identified after negative staining and appear darker...lentiviral preps.   Viruses are generated using standard methods that have been optimized for each specific...three fold higher than those achieved using the standard qPCR method.  Titering by the University of Pennsylvania...high quality viral vectors by optimizing and standardizing production protocols and performing rigorous...of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV subjected to ...production are propagated in the endA-mutated NEB Stable strain of E. coli. In addition, plasmids are typically...

...experiments and understand their data. Likewise, we want to contribute to helping others understand ours. Improving.... The PLOS policy on data access is a formal statement that is already practiced or required by many ... errors in assumptions, use of inappropriate statistical methods, omission of some data, etc.  The firestorm...published, this data is usually condensed into a statistically evaluated set of values.  However, to evaluate...sharable form is indeed a significant effort. Our standards of data recording are highly variable and usually... images are said to be representative or the statistical variation is calculated and placed in the legend...are used to publications presenting a polished, standard and usually limited view of data. In addition ...

...with physical map distances of 75 and 2 Mbp, with the calculated fluorescent distances correlating with...organization of the nucleus in space and time, and to understanding how nuclear organization varies across the life..., or how organization may be altered in disease states. dCas9 gets a colorful makeover Even without its...with the previously established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that...degree of chromatin compaction even for this small distance! To the authors’ knowledge, this work represents...applications Ma et al. developed this technique using standard fluorescence microscopy, and are thus limited ...priority, as researchers work to increase our understanding of chromatin organization. This fluorescent ...

...various stages in their careers to get advice on choosing a lab, picking your research topic, starting a lab... hard work, collaboration, and staying honest with personal goals. Stay tuned for the follow up blogs ... lab, and more. Before we start, we'd like to extend a special thank you to Dr. Tom Ellis (Imperial College...with full force. George Church 1. “Initial and sustained motivation is a huge component of success.” 2... "Choosing Your Research Project," “Advice for Starting Your Lab,” “Fun in the Lab!" and more.   Acknowledgments...Choosing a Research Project Advice for Scientists Starting a Lab ...

...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...

... on the variable domain and not so much on the constant, or Fc, domain. Sure, we all know that the Fc ... immune system's classical complement pathway, starting a cascade of events that results in the pores ...phagocytosis (ADCP) So far, our antibodies have induced “stabbing” and “poisoning,” but let’s be honest, the elimination... functions and all that antibodies can do! For instance, some Fc receptors, once bound to an antibody,...Fc receptors are intriguing from a therapeutic standpoint. Engineered monoclonal antibodies that can drive... and resources References Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate... Greenspan, N. S., & Casadevall, A. (2016). Ig Constant Region Effects on Variable Region Structure and...

...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...

...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...

...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...