...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...

...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...

...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...

...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...

...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 ...

...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...

...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...