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... a plasmid? Or how much time you have to spend cloning before you can start your experiment? What about...order? Sometimes, it feels like an eternity of cloning, waiting, and repeating before you can finally ...course, these were all 2018 numbers, and the intervening five years, inflation has occurred. Here at Addgene...in science – the time spent by the scientists designing and performing the experiments. This cost varies...and money across the board. Consider alternative cloning strategies There are many strategies beyond the...well) plate! How much do repositories help?   Designing precise calculations for any of the numbers in...Addgene Blog 5 factors to help you choose the right cloning method Find blog posts about scientific sharing...

... the tides may be turning. In 2013, the NIH announced its first round of Broadening Experience in Scientific...achieved tenure-track positions within 5-7 years of earning their PhDs (3), showing that the ‘standard’ pathway...Scientific Training (BEST) awards, which are designed to support new approaches that address the need to... School. Graduate education programs are also beginning to address these issues independently; for example... may look like. The panel discussion on Friday morning will be dedicated to Academic data and the Labor...PMC3342243. 4. Mathur, Ambika, et al. "Transforming training to reflect the workforce." Science Translational...

...visualization of both organic tissues and cell cultures, opening biological interiors to many inquisitive scientists...select the optimal dye or fluorescent ligand when designing an experiment. You may find yourself defaulting...Fluorescent dyes utilize these fluorophores by combining them with a molecular species with high affinity...affinity for an endogenous molecular target, meaning the dye will be attached to the target through binding...so extra care must be taken to assess if proper staining is achieved. Fluorescent ligands have more flexibility...) can be stably combined with nanoparticles, maintaining intensity at ~85% of its original value after...brightness and high degree of overlap. Dyes for DNA/RNA staining should have high affinity and specificity to nucleic...

..., I have conducted informational interviews with nine Addgenies. There are a few patterns throughout the... Graduate school: Do I or don’t I? I asked the nine Addgenies about why they did or did not go to grad... had always been a student and I truly enjoyed learning.” Addgene’s Director of Product Management Eric...feeling like I could read a scientific paper from beginning to end and really get it.” On the other hand, ...don’t get your PhD you’re set up for failure. Listening to Kenkel’s story has helped me realize that is... see where my life will take me. I’m also just learning. I have a huge interest in data science,” which...they work at Addgene. I posed this question to all nine of my interviewees. Fabienne Furt, Addgene’s Associate...

...maximum genetic heterozygosity in the population, meaning they more closely mimic the genetic diversity of...be engineered by inserting a targeting vector containing the gene of interest into a zygote, or into embryonic...microinjection of DNA or infection with a viral vector containing the gene construct can be used to insert the ...knockin mice. Co-injection of a targeting vector containing homologous ends to the breakpoint promotes homologous...engineering favors DNA repair via non-homologous end joining, the newly inserted gene may have errors introduced...creating the floxed gene segment. Crossing of mice containing a floxed gene to a Cre-expressing strain (which...post of this series, I’ll discuss crossing and maintaining a mouse strain. Additional resources on the...

...vector”. Cloning Method Choose from: Gateway cloning, gene synthesis, Gibson cloning, ligation-independent...ligation-independent cloning, restriction enzyme, TOPO cloning, unknown, or other. Bacterial Resistance Enter...possible, we propagate plasmids in the standard cloning strain DH5α. For plasmids with highly repetitive...plasmids, choose the NEB Stable strain. For plasmids containing the ccdB gene, such as Gateway vectors, choose...

...combined with. Combining a PSAM with the IPD of a cation channel, such as the serotonin 3 receptor, will...understand the complex wiring of the brain and the underpinnings of neurological disease, neuroscientists need...a popular tool in the field as it provides a noninvasive method for manipulating cell activity, especially...provided by chemogenetics are instead that it is noninvasive and has longer-lasting effects. Building your...issues can be mitigated by using low doses and running appropriate controls (Jendryka et al., 2019), they...promote depolarization in neurons. Alternatively, combining with the IPD of an anion channel, such as the ...

... nearly every experiment requiring AAV. Before joining my lab, I had never worked with AAV, so naturally... Two to five days after transfection, the AAV-containing cells and media are harvested and purified. In...duplicated regions like the ITRs. Restriction digest screening for ITR deletion is still necessary. To save time...overlaid. Following ultracentrifugation, the AAV-containing 40% fraction is collected and buffer exchanged...titering by qPCR quantifies the number of genome-containing viral particles that are present in an AAV prep...standard curve, which is generated from an ITR-containing plasmid. This whole process takes ~3 hours to...expression in mammals Use the PEI calculator for planning AAV packaging transfections Find tips for a first...

...you should always ensure nothing is covering it. Turning on the UV light 3-4 times a week for up to half...microbe-laden aerosols that may enter the hood. Planning your experiments thoroughly before entering the...solves most problems before they occur. With proper planning, you’ll know exactly what materials you’ll need...changes in temperature (especially if you have air conditioning on) so leave them in a sterile incubator. This...make up your master stock, while you can use the remaining vial to grow and freeze additional cells that ... Universität Berlin, Germany. She will soon be joining her PhD lab at the Max Planck Institute of Colloids...the Addgene Blog The Challenges of Cell Culture Cloning Mammalian Cells with the Agarose Method Plasmids...

...were used in this study.  The zCas9i cloning kit is a plant cloning kit that contains a series of vectors...Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple antibody-based vaccines...cross-reactivity, antibody candidates underwent affinity screening to select for slow off-rate binders. The best ...protein VP1 is co-expressed with a fusion protein containing VP2C and a protein of interest (POI). The VP2C...with more on the way. S5E2 regulatory element containing vectors developed in the Jordane Dimidschsteind...provide expression selectively in Parvalbumin containing cortical interneurons. We now provide ready-to-use...

...Neurological Surgery at the University of Miami. The beginning of this century has seen some major advances in...originally named orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David Burns and Francis...confocal microscopy In confocal microscopes, optical sectioning of a specimen is based on discriminating the ...to photobleaching.  Furthermore, point-by-point scanning along the entire specimen makes imaging too slow...photo-bleaching or photo-damage than in conventional laser scanning microscopy . Since image acquisition is performed... of imaging rodent brains and spinal cords by combining Ultramicroscopy with tetrahydrofuran-based tissue...Leischner, A. Schierloh, N. Jährling, C.P. Mauch,  K. Deininger, et al. (2007). Nature Methods, 4, 331–336. Pubmed...

...Recently I learned that Addgene’s pLKO.1 cloning protocol is viewed around 3,000 times each month. I ...Addgene website in December 2006, the pLKO.1-TRC cloning vector (deposited by David Root of the Broad Institute...this plasmid, and if it still being used. After learning of it's continued popularity, I decided this protocol...scientists, publishers, and small start-ups are turning to software apps to solve our need for quicker,...protocol listed under “Supplemental Document”: gRNA_Cloning Vector (deposited by George Church’s lab). Addgene...has also cataloged a variety of useful molecular cloning protocols, including making LB agar plates, restriction...labs, and now Addgene. I just began uploading our cloning protocols to the site and will keep you updated...

...loss of function meaning that if an essential gene plays a role in the desired screening phenotype, it may...knock out, inhibit or activate target genes by combining specific sgRNAs with Cas9 or Cas9 derivatives....copy number dsDNA cutting. A Dolcetto library containing only 3 sgRNAs (setA) also outperformed CRISPRi...conducted with only two constructs (the other containing dCas9-VP16). Like CRISPRi, CRISPRa is only effective...the SAM library approach in a comparison study screening for vemurafenib resistance in A375 cells. The ...screens Read this blog post about genome-wide screening using CRISPR Resources on Addgene.org Find CRISPR...

...so that you can specifically isolate bacteria containing your constructs of interest. But does it matter...will lose it during division. These non-plasmid-containing bacteria can compete with and confound the goal...from forming while effectively selecting plasmid-containing bacteria. So… why do some people still use amp...? Zeocin is genotoxic when not 100% inhibited, meaning it will induce mutations in the host/plasmid DNA...Excellent No Bacteria, plants, and eukaryotes Not Tn5-containing cells {% icon icon_set="fontawesome-5.14.0" name...References and Resources References Hwang, J. M., Piccinini, T. E., Lammel, C. J., Hadley, W. K., & Brooks...Antibiotic Resistance Plasmids 101: Blue-White Screening Choosing Your Perfect Plasmid Backbone  ...

...research interests include proteomics, machine learning, and visualization for big biology. GPS for the...1, 10) indicates a stretch of DNA at the very beginning of the third chromosome, ten base pairs in length... us to transform raw genomic coordinates into meaningful experiments. Table 1: An emerging family of "...all of those 13,000 regions are important for maintaining ESCs. So which of these 13,000 regions are critical...enhancers! This leads us to a three-step approach for designing an experiment to identify the critical NANOG binding...Lentivirus from Addgene If you’re interested in designing your own CRISPR screens for enhancers, check out...database. Aneesh's research interests span machine learning, proteomics, and user interfaces for math.     ...

... on the Society for Laboratory Automation and Screening (SLAS) Electronic Laboratory Neighborhood.   Work...If you enjoy working and come home happy in the evening, your friends and family will truly sense and appreciate...within these bounds You can work later (in the evening, or in a few years) when life responsibilities ...Someone else can clean your house, pick up your dry cleaning, cook your dinner and deliver your groceries. ...nights when you can’t get home, babysitting or running errands. Give help when you have time, and in return... things can’t. I kiss my kids good night every evening I am home no matter how busy I am. Believe it or... really busy, but I go to the gym almost every morning. Of course, this is more than just fun, as your...

...letter, they have an opening, a body, and a closing. Parts of a cover letter The opening paragraph is the... by giving specific examples of your work and explaining how your experience and skills would help you...application. These could be logistical, like briefly explaining a gap in your job history or sharing relocation...your organizational system, your rationale for designing it the way you did, and state that it would help...how you feel connected to their work. Since the beginning is often the hardest part, here are some examples... in [specific topic] and in the possibility of joining your team as a postdoctoral researcher. Dear Hiring...

...after negative staining. Empty vector particles can be identified after negative staining and appear darker...Physical titer: the concentration of viral particles containing viral genomes. Physical titers are measured by...full to empty viral capsids: the ratio of genome-containing viral particles relative to the total number ...approximately 50% of viral capsids were genome-containing (“full”) (Zeltner et al., 2010), while another...Grimm et al., 1999). The percentage of genome-containing viral capsids is typically quantified by electron...representative viral vector preparations are genome-containing (Figure 1).    Figure 1: Electron micrograph...

...muscle expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into kynurenic acid...muscles led to another clue: differences in the kynurenine pathway of tryptophan degradation. It turns out... acid. That’s key because, unlike kynurenine, kynurenic acid can’t cross the blood-brain barrier. By lowering... lowering circulating levels of kynurenine in the body, the brain is protected from stress-induced biochemical...References: Skeletal muscle PGC-1α1 modulates kynurenine metabolism and mediates resilience to stress-...

...plasmids String assembly gRNA cloning (STAgR) String assembly gRNA cloning (STAgR), developed by Stefan...Read the paper in Genome Biology Find plasmids containing these base editors Spatially and temporally...gRNA multiplexing system that generates vectors containing high numbers of gRNAs in one overnight reaction... Read the paper in PLoS One Find the STAgR cloning plasmids Mobile-CRISPRi allows CRISPRi experiments...Cas12a inhibitors Using a bioinformatics and a screening approach, the Jennifer Doudna lab identified three...