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...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...

... above. A trained machine learning algorithm was able to detect anthocyanin even when it was not obvious...could equip plants with a way to warn us about concerning growing conditions? That was the goal of Dmitri...purple when they detect a specific molecule. By combining this approach with receptors for problematic ligands...Center.  The plants’ purple comes from an anthocyanin, an endogenous pigment typically produced as ...identified the genes required to activate the anthocyanin synthetic pathway and assembled them into a synthetic...

...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...

...introduced what’s been called a major breakthrough in cloning technology with the Golden Gate method. For the...researchers working in plant model systems. “The cloning is all done in vitro; it’s no different in mouse...to more tedious and time-consuming traditional cloning methods, Lohmann said. The key component of Golden... says their lab learned the hard way after the cloning method, which had been humming along at 99 percent... "GreenGate - A Novel, Versatile and Efficient Cloning System for Plant Transgenesis." PLoS ONE. 8(12)... Engler et al. "A One Pot, One Step, Precision Cloning Method with High Throughput Capability." PLoS ONE...

...dynamics in live mice under a variety of behaviors, opening up new research directions that were inaccessible...concentrations in cells than existing sensors. Using genome mining and structure-guided engineering of potassium binding.... Figure 5: Structure-guided design and genome mining both led to new sensors with lower binding affinities...sensor plasmids here!   Torres Cabán CC, et al. Tuning the Sensitivity of Genetically Encoded Fluorescent... Indicators through Structure-Guided and Genome Mining Strategies. ACS Sens. 2022 May 27;7(5):1336-1346...cellular concentrations of lactate. The microfluidic screening platform developed here is also a promising approach...Figure 6: A) Schematic of BeadScan platform for screening candidate biosensor variants for performance under...

...however, inconvenient data must not be ignored. Returning to an earlier analogy, no amount of shouting will...scientists have chosen to leave the lab and march. Unintended consequences While frustration and anxiety have... science, others are concerned about possible unintended consequences of a march. Three of these risks...of everyone in government (19). To defend the meaning of the word ‘fact’. Policy must be based on our...science advocacy opportunities. Information for joining the march: March for Science: The website for...For science to be integrated into politics in meaningful ways, continued conversation about science’s ...: Universities Report Fourth Straight Year of Declining Federal R&D Funding in FY 2015 National Science...

...useful kits you can order from the repository: FX Cloning Kit BIOFAB Plasmid Set pCri System Cell Free Protein...please write to us in the comments section.  FX cloning kit This kit is designed to be used with Type IIS...sites used in these vectors are of low abundance (meaning they are unlikely to be found in your gene of interest... incorporation of extensive sequences used for cloning into the final translated products. Negative selection... help decrease false positives observed in the cloning process, making it easier to produce the constructs...interest in a variety of model systems, thereby opening your research to a variety of experimental opportunities...fusions all use the same restriction enzymes for cloning. Furthermore, some plasmids contain peptides to...

...compared to other delivery methods. Retrovirus has a cloning capacity of ~8 kb, so either a single polycistronic...dividing and nondividing cells and have a larger cloning capacity (8-10 kb) than other retroviral vectors...between a vector and chromosomal DNA. PB has a cloning capacity of ~9-14 kb. This system is transfected...transfected into cells as a donor plasmid containing the transposon, along with a helper plasmid that expresses...which is removed by culturing cells at 38℃, but screening cells for the presence of transgenes is still ...help mediate their transduction, such as the polyarginine peptide described in Zhou et al and Kim et ...large nucleoli, and little cytoplasm. Positive staining for alkaline phosphatase, expression of embryonic...

...appropriate choice (Panina, 2018). Look for studies specifically aimed at determining the best options for...Zhang M, Hu G, Fang F. Fudenine, a C-terminal truncated rat homologue of mouse prominin, is blood glucose-... amounts of reagents in your chemistry, unless running a multiplex reaction or on a rapid cycle machine... quick way to validate your primer probe is by running your qPCR product on an agarose gel, then cutting...real-time RT-PCR. J. Immunol. Methods 283:291–306.Panina, Y., Germond, A., Masui, S. et al. Validation of...Digital Droplet PCR for AAV QuantificationPlasmid Cloning by PCR...

...contain BsaI sites for easy cloning of a gRNA of interest. Cytidine and adenine base editing in Streptomyces...breaks are lethal because the non-homologous end-joining (NHEJ) repair pathway is not very robust. In many...applications for which they’ve been used. The beginnings of bacterial CRISPR engineering One common way...template, using Gibson Assembly or restriction enzyme cloning. Streptomyces bacteria are more recombinogenic ...directly modify a given gene. CRISPR-tranposons By combining CRISPR editing and transposons, the Sternberg ...inducer, in this case anhydro-tetracycline (aTc). By cloning in two tandem copies of the gRNA cassette, one ...Sun lab demonstrates the utility of cytidine and adenine base editors that enable C-to-T and A-to-G base...

...bystander editing by adenine base editors (ABEs). In base editing, bystander edits are unintended changes to nearby...mostly or entirely replaced with DNA bases while retaining activity in both Cas9 and Cas12 (Kim et al., 2020... guides are unlikely to impact other kinds of unintended edits, like guide-independent deamination by ...Improved specificity and efficiency of in vivo adenine base editing therapies with hybrid guide RNAs. ...Cas9 Structural Biology CRISPR 101: Cytosine and Adenine Base Editors Additional resources on addgene.org...

...expand my knowledge more than ever before. Before joining Addgene, I had spent most of my junior year stressed...well as complete the lab safety and cybersecurity training before stepping foot into the lab itself. I appreciated... as equals, because as soon as I had passed my training, I dove straight into my capstone project. A capstone...wrong. My capstone project was based on plasmid cloning and I, the researcher, got to choose what gene ... winner. We used the next 4-5 weeks to work on cloning the Syntaxin3 gene in a pLenti vector backbone....assembly but decided to go ahead anyway. We were running on a time crunch and knew that if we were to restart...

...up the Addgene booth on Thursday evening, I stayed to see the Opening Keynote address with Bill Gates.... lab). Bright and early on Saturday morning, John Doench’s opening presentation at the CRISPR screens ...well-optimized CRISPR screening technology. Addgene got several shout-outs for making popular screening libraries ...

...you need until the team really starts functioning (or malfunctioning). Clear lines of communication Few ...that many researchers feel that they lack formal training in a variety of transferable skills. At Addgene...The famous discoveries and experiments I’d been learning about for years were usually the products of teams...I can also orchestrate the formation of high-functioning-but-temporary teams that have a specific purpose...snapped out of the phase of my life that had me listening to Simon & Garfunkel’s “I Am A Rock” on repeat...a team is a really good way of creating and strengthening those social bonds. So never mind how much better...

...If you’re interested in learning a new skill, getting access to equipment you don’t have, or maybe both...researcher remains employed by their home institute, meaning the host university does not hire or pay them (...current institute. A common reason is hands-on training in a technique that only a few labs have expertise...this expertise will provide you with guidance and training so that you yourself can become an expert and ... Identify reagents you will need, any required training, and a priority list of experiments you hope to... Visiting scientist positions take commitment, planning, and funding – they may not be right or necessary...

...FedEx envelopes in a single day -- all while maintaining social distance.  From front to back: David Gordon... fight against coronavirus We are just at the beginning of understanding how SARS-CoV-2 interacts with...used HEK293T cells in our first study, but we're planning to soon expand to other cell types, including ...community's help. You could be the next scientist joining in this worldwide fight against SARS-CoV-2. As ...flanked by EcoRI and BamHI restriction sites - so cloning projects should be fun and straight-forward. All...confirmed expression via mass spec for two of the remaining viral proteins. Only Nsp11 ran bigger than expected...

...PMID: 30704911. Acoustic reporter genes for noninvasive imaging of microbes in mammalian hosts Article...podcast segment In recent years, scientists are beginning to find connections between the microbiome and...largest miRNA sensor collection reported to date, containing all 620 sequences of probable mature human miRNAs...consists of a library of reporter plasmids, each containing two fluorescent proteins and four repeats of ...plasmids is amenable to Golden Gate assembly for turning the single-input reporters into multi-input cell...al., Nat Commun, 2019. PubMed PMID: 29934631. Screening immune cell lineages in vivo using the CRISPR-...

...component. Alternative screening techniques While CRISPR and drugs dominate the screening scene, there are ... a selection, you might put cells into a drug-containing media in which only cells which can tolerate ...is typically not feasible to test in any given screening experiment. Thus, a limited panel is usually selected... sensitive enough? No one wants to spend time screening hits that aren’t “real” or novel for their purposes...Dijkhuizen, L. Directed evolution of enzymes: Library screening strategies. IUBMB Life, 61: 222-228 (2009). doi...

... all scientists? Curiosity? Passion? A love of learning? No. Or at least not entirely. What truly unites...the Issue You can’t fix an experiment by simply running it unchanged over and over again, and the same ...works, so do it anyway.  You may find that after running a protocol a few times, you no longer need the ...few key reminders on a sticky note   Visual Planning  Plate maps, pre-labeling tubes, and even drawing...stressed, or rushing. I would spend the time quietly running through the experiment in my head, double-checking...another short "quiet period" instead of immediately running to the next thing.  It may seem counterintuitive...