...fluorescent biosensor has a high sensitivity (50 nM-500nM range) and a high specificity for SLs in vitro...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...organoids. The Organoid EasyTag System is a straightforward workflow for doing gene targeting, reporter.... This novel type of biosensors could be used in high throughput screening for agrochemical compounds ...CRISPR Plasmids and Resources page. Here are some highlights from the past couple months: A new lentiviral...

... the high school interns as equals, because as soon as I had passed my training, I dove straight into ... never would have imagined that my summer before high school senior year would have been spent within ...weeks that we had made a mistake by not having the right concentration of a reagent during our HiFi assembly...the following two weeks, we were able to obtain eight clones. We ran digested and undigested samples of...one clone we found with the correct bands was the right plasmid, so after discussion, we sent it for sequencing...invited guests came to see the research from all the high school interns, done within the past six weeks. ...other way. Toj Toj-Villagran is a rising senior in high school who just completed their summer internship...

... tips for high efficiency cloning with this technique: Amplification efficiency is highest when using...labs, a variety of other cloning techniques with higher efficiency and simpler procedures have been developed... in the figure from Chaokun et al., 2011 to the right. After PCR, you should confirm that the products...introduced during PCR or any subsequent steps. Using high fidelity DNA polymerases, I have never had any mutations...chance of introducing an unwanted mutation. Tips for high efficiency cloning Now that you have a better understanding...using high fidelity DNA polymerases like phusion. An 18-cycle PCR leads to the most colonies after transformation...27391134. 2. Li, Chaokun, et al. "FastCloning: a highly simplified, purification-free, sequence-and ligation-independent...

...neurons in the mouse brain. MouseLight images can be found on the MouseLight website. Together with scientists...neuroanatomy and neural connectivity, providing valuable insights for neuronal communication. Viral vectors and...vectors can be equipped with strong promoters for high and neuron-specific transgene expression. Fluorescent...for their production and purification that yield high titers (see also AAV protocols for production, purification...researchers from the Janelia Campus for their MouseLight project but neurons were traced with AAV2. Two-Photon...variant, rAAV2-retro, that migrates retrogradely at higher rates than any other AAV serotype (Tervo et al....also having the added advantage of lower toxicity, higher specificity and reduced technical variability compared...

...forward, in this post I’ll point out some of the highlights and takeaways I got from the symposium. The ...outcome is not easily achieved. Of note, it was highlighted during the conference that just increasing funding...projects sought to make an interactive game that highlights just this point. As part of this, trainees need...thoughts The FOR Symposium Boston 2015 shined a light on some of the many problems facing the academic...academic research enterprise. In addition to highlighting some of the important questions that still need to...

...with different properties (for example, overall brightness or on/off kinetics) that makes them suitable ...(pGP-AAV-syn-FLEX-jGCaMP7b-WPRE) exhibits the brightest resting fluorescence of the GCaMP7 family of calcium...pAAV.hSyn-FLEX.iGABASnFR) has good membrane localization and brightness. The F102G variant (pAAV.hSyn-FLEX.iGABASnFR....through imaging. Since it’s illuminated by red/orange light, Archon1 can also be used in conjunction with optogenetic... activators and inhibitors that respond to blue light. We recently released a constitutive (pAAV-Syn-Archon1...-jGCaMP7f-WPRE AAV1 with a CAG promoter exhibits high specificity for Purkinje neurons. 105321  AAV1...pGP-AAV-syn-jGCaMP7c variant 1513-WPRE 7c exhibits high contrast between peak fluorescence and resting fluorescence...

...Some common ones you might see include ColE1, pMB1 (which comes in a few slightly different but well known...make additional copies. ori sequences are generally high in As and Ts. Why, you ask? Well, A-T base pairs...make a lot of your plasmid? Is the gene toxic in high amounts? It is always good to keep in mind that ...propagate plasmids, but endA- E. coli are best for high yields of plasmids. Growth conditions The amount...culture inoculum Freshly streaked bacteria have higher copy numbers – for optimal results always pick ...cultures. Freshly transformed cells also give a higher plasmid yield that colonies streaked from a glycerol...stock. Incubation for 12-16 hours tends to give higher copy numbers since the bacteria have just reached...

...While I had always enjoyed learning about biology in high school, it wasn’t until I started working in my ...molecular biology-based activities making their way into high school classrooms, like gel electrophoresis to separate...led me to develop BioBits ExplorerTM and BioBits BrightTM, biology kits based on freeze-dried, cell-free... that students can easily sense, whether that’s bright fluorescent proteins that they can see, enzymes...initially developed the BioBits kit to be used in high school classrooms, but it could be widely adapted... a biology teacher at the elementary, middle, or high school level, feel free to check out one of our ...PMC6070312. 2. Stark, Jessica C., et al. "BioBits™ Bright: A fluorescent synthetic biology education kit....

...projects in many labs. However, the high costs of sample preparation for high-throughput next generation (NGS...Addgene to develop a highly optimized bioinformatics pipeline that will return high-quality assembled and...quantities of library molecules for each sample remain highly normalized across a wide range of input DNA concentrations... vary by DNA input amount. plexWell™ + Addgene = high quality NGS-assembled plasmids Because of the enhanced... plasmids. The plexWell™ workflow also offers a highly efficient NGS-based alternative to Sanger sequencing...assembly pipeline. To date, this has resulted in high quality NGS-based assemblies for over 3000 plasmids...

...fluorescence changes or a requirement for very high excitation light intensities have made these indicators difficult...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...measure ApoB-LPs directly and develop a potentially high throughput in vivo model to identify drug candidates...Thierer et al, 2019). Zebrafish are a great model for high throughput study of ApoB-LPs because their ApoB ...amenable to use with other bEBP/UAS pairs, and displays high dynamic range. However, the promoter in question...lipid compositions of C. elegans membranes, which might beneficially influence the rhodopsin properties....

...commonly used, PVDF tends to be slightly more popular due to its durability and high binding capacity. In addition... (HRP), that emits light upon reacting with a specific substrate. The emitted light, or chemiluminescence... samples, a protein standard of known molecular weight in a separate lane can help confirm the size of...acrylamide content of the gel. For small proteins, use a higher percentage of acrylamide to increase migration ...broad range of protein sizes but takes hours to overnight.  In contrast, a semi-dry transfer only requires...chain. Consequently, antibodies validated for IP might not work for a western and vice versa.  In addition...specific experimental condition you could look for a slight size shift in the protein band before and after...

...Antibodies 101: Choosing the Right Isotype Antibodies 101: Selecting the Right Antibody Resources on Addgene.org...groups on a western blot and look for the molecular weight shifts indicative of tau phosphorylation. By including...protein standard (P) to check for tau molecular weight shifts. A negative control is a sample or ...runs. If the results vary considerably, then the high degree of variability in the process could make ...signal from your target needs to be significantly higher than the background noise (Waters 2009). If the...the fluorescence from your target is extremely bright, then the noise likely will not interfere. If the...control for all new samples, protocol changes, or when high background labeling occurs (Burry 2011). Complex...

...junior faculty member at Harvard Medical School/Brigham and Women’s Hospital (BWH). Based on her experience...years ago looking for their career improvement) and bright students that will be the future of the country...to help build, together with the universities, a bright future. IMP provides matched one-on-one international...Although all mentorship relationships are valuable and highly recommended, a mentor with international experience...impressions of the IMP experience. Conference highlights included remarks by the Spanish Secretary of ...representatives from different Spanish universities and high level public and private institutions also showed...website Learn about the Mentor Circle Program at Brigham and Women's Hospital Visit the International Mentoring...

... be on LinkedIn?” For anyone who is planning or might need to plan for a career outside academia (that...business or follow up. Use the "Contact info" link right below their photo. 2) Personalize all connection...note, you waste an opportunity to start things out right. Also, many people on LinkedIn don’t appreciate ...connect button unless you’re on a profile page. You might be looking at a list of suggested connections and...connect people that want to meet or that you think might be helpful to each other. This is a quick way to... before you’ve had a chance to proof read. This might not be important for a good friend, but if you are...and it’s still connecting with a stranger so is highly discouraged. You should be using your network to...

...catalog to help you find the right split-FP plasmid for your experiment.  Highlighted Split-Fluorescent Protein...bimolecular fluorescence complementation assay with a high signal-to-noise ratio. Kodama Y, Hu CD. Biotechniques...amino-terminal acetylation system and novel in vivo high-throughput screen for molecules affecting alpha-...Improved sfCherry3C(1-10) for use with sfCherry2(11) Bright split red fluorescent proteins for the visualization... chromophore formation (abundant in eukaryotes) Bright monomeric near-infrared fluorescent proteins as... with exogenous fluorophores, which can even be bright enough for single-molecule tracking in live cells...

...PAM) might work too.  High dCas9 and gRNA expression are key for complete DNA demethylation – tight binding...the Future, and big hair. To what was perhaps a slightly smaller population, something else was also suddenly...conference or a karaoke bar), you probably want to sigh. The problem is that many of these studies report... 2018). This means that off-target loci – in neighboring chromatin or throughout the genome – could be...reality, editing your target site is not as straightforward as expected. TET1 can have many effects independent...and pLM-CMV-R-Cre. Selection for cells stably and highly expressing these constructs can be achieved by ...

...applications and gene therapy: they can be produced at high titers, can accommodate large transgenes, transduce...Gutless Ad vectors (helper-dependent (HD-Ad), or high-capacity (HC-Ad__ are devoid of viral genes and ...belongs to this category. 3rd Gen AdV (Gutless or high capacity AdV) are devoid of all viral coding sequences...proteins in trans. The Gutless AdV have generated high interest for gene therapy due their increased cloning...more HEK293 cells and produce more rAdV to reach higher titers. Successfully infected cells will become...separate rAdV from other cellular debris. Purified high-titer rAdV is then dialyzed and stored at -80C. ...account. You're now ready to use your purified, high-titer rAdV in your experiment. Enjoy! References...

...midst of a crisis. The “antibody crisis” came to light as antibodies became commercialized, revealing the...goldilocks antibodies. A goldilocks antibody is highly specific (minimal binding to off-target molecules...creating a public and accessible database for low-cost, high-quality antibodies for IHC. Working with collaborators...their recombinant forms (rAbs). mAbs and rAbs have higher homogeneity than polyclonal antibodies because ...from the negative control, such as showing a very bright signal only in the hippocampus compared to other...the amount of time the sample needs to be under light to discern the signal. If the exposure time is long...allowing scientists to find and utilize reliable, high-quality antibodies. We are creating a database for...

...injection site is straightforward, as it targets large muscle groups (usually the thigh), though it is important...injection, like you might after getting a vaccine. Restraint: mechanical restraint Location: thigh Gauge: 25–27...use depends on a number of factors. You have to weigh the pros and cons of various methods and choose ...circulation is slower, and more institutional oversight is required. Restraint: mechanical restraint or...such as experimental therapies or pain relief, straight to the stomach. This technique involves administering...Intermediate Intramuscular Mechanical restrainer Thigh 25–27 < 0.02–0.05 mL Beginner Subcutaneous Back...

...emission spectra, meaning they will emit fluorescent light at similar wavelengths (See this handy emission ...against APC-Cy7, the populations are in more of a straight line and are only positive for APC (Figure 1B)...horizontal line, so our circled population on the right needs to shift towards the X axis to become a perpendicular... plot in 2A, the populations are not perfectly straight, but the center of each population is in a line... sample you are trying to compensate in the top right corner of a flow plot, meaning that it should be..., it will show up as a diagonal line in the top right corner of the plot and large compensation values...positive sample. The cell population in the top right corner of the plot is positive for both markers,...