...Topoisomerase I is released from the vector when the PCR product and vector ligate; however, it can potentially...this sequence. The energy from this breakage is stored as a covalent bond between the cleaved 3′ DNA strand... days commercially available TOPO kits provide vectors or cloning arms with overhanging 3´ deoxythymidine...residues that are covalently linked to topoisomerase. Vectors in these kits also often have the topoisomerase...screening after transformation - self joining of the vector ends results in the production of blue colonies...Reaction: Mix together the PCR product and TOPO Vector.  3. Incubate 5 Minutes at Room Temperature: You...are planning to transform right away OR you can store the reaction at -20C overnight. 4. Transform TOPO...

... introduced, stochasticity will result in a combinatorial expression of these three genes with different...inherently low. This is because the stochastic and combinatorial expression of fluorescent proteins is only possible...Tetbow In modern neuroscience, plasmid or viral vector-mediated gene delivery is more common than conventional...conventional transgenic strategies. In such vector-based gene delivery systems, the stochastic expression...original method, we used the Tetracycline trans-activator system to boost the expression levels, and we ...we named the system Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018) (Figure 1)....the functional and anatomical maturation of the olfactory system.      Takeshi Imai is a professor at Kyushu...

... plasmids in the repository, each verified by sequencing, which makes the repository a convenient source...content and rare codon percentage were not good predictors. There is not much variation in GC content between...for GC content. Rare codons also are not good predictors because they are just that: rare. “Nature has...says. Percent sequence identity was a different story - the team found that the percent sequence identity... a synthetic version of that gene was a clear predictor of whether the gene is synthetic or natural. They...most common expression system within the Addgene repository is mammalian, but the largest source of unique...is from Proteobacteria to mammalian expression vectors. And more broadly, their data revealed that the...

...upstream of an effector/reporter gene. In cells where the stop cassette is removed, the effector is expressed...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...cells with sgRNAiBAR library containing lentiviral vectors the iBARs can be detected by next generation sequencing...including Drosophila, fish, and mice, by replacing the effector with a gene of interest in the cells you are studying...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ...screening, calcineurin dynamics measurements, or monitoring in vivo. To overcome these technical issues, ...4X CDRE repeat. In addition to showing that this vector is suitable for flow cytometry and fluorescence...

...co-founder Melina Fan and former Associate Director of Viral Vectors Karen Guerin for the journal Molecular... in Gene Therapy, Director of the Grousbeck Gene Therapy Center, Associate Director of the Ocular Genomics...Addgene welcomes three new members to the Board of Directors: Khandan Baradaran, Frank Stegmeier, and Luk Vandenberghe...Baradaran’s expertise in viral safety, gene therapy vector development, vaccine development, and assay technologies...funded,” she says. Baradaran met Addgene Executive Director Joanne Kamens several years ago through the Healthcare...Officer at KSQ Therapeutics and is an Addgene depositor. KSQ uses a CRISPR-Cas9 platform to systematically...yeast and did a genetic screen to identify the factors that regulate cell division. And that really got...

...continuously monitor multiple systems. Ideally this tool would be wireless, able to monitor outcomes of...importantly distortion of the fibers didn’t affect their function (the gut twists and contorts during the...probe due to the lack of tools to continuously monitor it. How to address this? Through the pairing of...Previously, there were very few tools available for monitoring how both the brain and visceral organs interact...harvested after the behavior, or the animals being monitored were physically hooked up to invasive, cumbersome...indicating the system was indeed stable for continuous monitoring. On the gut side of things, the authors optigenetically...studies Channel rhodopsins for optogenetics Viral Vectors 101: Chemogenetics ...

...in a mammalian expression vector (pcDNA3) and a bacterial expression vector (pRSETB). Fabritius A, et...achieve this they created a lentiviral vector system that monitors both APOBEC- and Cas9-mediated Editing...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...blue light. This tool has now been modified by Torsten Wittmann’s lab at UCSF to modulate cellular dynamics...New tools for developing new FPs - the mCarmine story Article contributed by Caroline LaManna  Listen ...segment The increasing popularity of single base-editors has resulted in the rapid expansion of the genome...rapid fluorescence-based quantification of base editors that utilize dCas9 fused to cytidine deaminases...

...deaminase-mediated base editing (cytosine base editor or adenine base editor) and reverse transcriptase-mediated ... et al., 2019). Both cytosine base editors and adenine base editors have been developed for plants and...CRISPR has taken the genome engineering world by storm owing to its ease of use and utility in a wide variety... al., 2013). Nuclease dead Cas9 (dCas9) based activators (such as dCas9-VP64) or repressors (dCas9-KRAB...variety of Agrobacterium-compatible, Cas9 containing vectors ready for Golden Gate mediated cloning of your ... the Patron Lab.  As with all plasmids in the repository, we highly recommend reading the associated publications...Z, Wang Y, Wang X (2019) Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper...

...molecule integrin inhibitors and natural ligands, such as fibronectin, to block receptor function, e.g.,...technologies and laboratory techniques related to antibody discovery. You can find cell surface receptor biology...Integrins are essential cell surface receptors involved in complex signaling pathways and the linkage...Despite their ubiquity and significance, these receptors have proved challenging to study. Cells often ...integrin antibodies available through Addgene’s repository. The collection includes 26 unique antibodies...from its closed to its open state, activating the receptor. Ligands can bind to integrins in their closed...subunit. Using these affinity reagents, the investigators inhibited other RGD-binding integrins, individually...

...characteristics that make them ideal for studying in a laboratory environment. We’ve covered five model organisms...are five more model organisms that have left a historical impact on the scientific community. (And, check...have been specifically developed to thrive in laboratory settings. One of its most attractive features...Level 1 microbe makes it a perfect candidate for introductory teaching and research environments (Idalia &...  The E. coli genome has an extensive research history and characterization. Many proteins and processes...studies. Elly Tanaka’s lab has constructed several vectors for use in the axolotl including plasmids for use...organisms in the world. With its extensive scientific history, highly characterized genome, and widespread utilization...

...contributed by guest blogger Natalie Niemi, a postdoctoral fellow at the Morgridge Institute for Research...phosphorylation sites. If knowledge of a candidate regulatory kinase is known, it’s possible to generate non-phosphorylated...Often, however, researchers lack knowledge of the regulatory kinase, access to active kinase for the reaction...tRNA with phosphoserine A specialized elongation factor that allows proper extension and translation of...synthetase, and EF-Sep – an optimized elongation factor that accepts Sep-charged tRNAs during translation...This modification allowed the deletion of release factor 1, the protein responsible for terminating translation...TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing GFP that can ...

...the abundance of a protein variant library is monitored by fusing the variants to eGFP followed by an ... is an enzyme critical for activating clotting factors and is the target of the anticoagulant drug warfarin...serotonin sensor The goal of Elizabeth Unger’s postdoctoral work in Lin Tian’s lab at UC Davis is to generate...serotonin is a well-known but poorly studied neuromodulator, and evidence suggests that it is incredibly...sensor). In preliminary studies with the lab’s collaborators, they were able to use the serotonin sensor ...activity. Unger also plans to use the serotonin receptor to study how single nucleotide polymorphisms present...and dose response. Selective serotonin reuptake inhibitors are frequently used to treat depression and anxiety...

...upon a moratorium until another meeting, three branches – a private funder, government regulators, and ...there was a generally agreed-upon international moratorium on utilizing CRISPR/Cas9 in humans. This topic...mtDNA transfer options being investigated; A UK regulatory body tasked two separate non-governmental groups...technology to research centers engaged in long-term, mandatory follow-ups and observational studies of the technique...whether or not scientists can recognize so-called bad actors among them and “self-police” or whether the media...to argue otherwise). To be sure, throughout the history of medical research, there have always been people...stop punting decisions on scientific, ethical, regulatory, and general public consensus to the next meeting...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...activation, using expression of differentiation factors as the screen readout. This retroviral mouse CRISPR...library can be used for screening of cells that are refractory to lentiviral transduction, now enabling forward...lipoproteins, shuttles lipoproteins through the circulatory systems, but can also lead to the formation of...as both a powerful tool for generating complex regulatory systems and further the study of natural σ54-... PMID: 31451697. Genetically encoded voltage indicators for use in worms Article contributed by Brook...Brook Pyhtila Genetically encoded voltage indicators (GEVIs) change fluorescence in real time when neurons...

... fields, including synthetic biology. Addgene depositors Christopher Voigt and Jeffrey Tabor have been...and inactivate biological processes. Chemical effectors are suboptimal for a number of reasons, including...cross-reactivity with other pathways. Since these effectors are diffusible, they’re also not suitable for ...histidine kinase that phosphorylates a response regulator (RR). TCS-induced responses are varied; one outcome...conditions, the chromophore activates the response regulator via phosphorylation, promoting transcription of...spread across three plasmids, with the response regulator encoded in the genome. To make the system less...The utility of these TCSs was limited by a few factors. These bulky systems were spread over a number ...

...effect of mutations of these regulatory elements on gene expression. The regulatory element (wild-type or mutant... from live cells. Monitor miRNA knockdown Expression of luciferase can be monitored as a downstream measurement...gene, such as luciferase, usually serves as an indicator of transcription within cells, where detection...Kinetics of Photon Generation Slower Faster Cofactors/Substrates Required Not Required Signal Strength...Organism Peak emission (nm) Size (kDa) Substrate Cofactors Secreted Luminous shrimp(Oplophorus gracilirostris... most often used to investigate the effect of regulatory elements, such as promoters, enhancers and untranslated...can then be used to quantify the effect of the regulatory element. Depending on the specific luciferase...

... important, factors to consider when seeking your dream lab. The principal investigator's point of view... hires are simpler because of their documented history in science. Potential graduate students typically...goals and chat about awesome science. Important factors in the search for a lab Studies show that job satisfaction...both work style and development needs. These two factors will provide a solid foundation on which to thrive... this should (in most cases) not be the driving factor. The following questions can serve as a guide for...you take the position. Am I choosing the right mentor? Make sure your advisors will be good role models...more on this topic see our blog "Choosing a Good Mentor for Scientists". This post also includes a useful...

...project management. Mentorship is another invaluable resource: connecting with mentors who have made similar...management often means stepping into unfamiliar territory. Unlike solving technical problems in the lab,...lab, management involves a different skill set: mentoring, setting strategic goals, and fostering team success...for me. I began exploring alternative career trajectories within the industry, but the transition wasn... role is also crucial. I owe quite a bit to my mentors, as they encouraged me to explore small-scale opportunities...company by taking on responsibilities such as mentoring a junior colleague. Guiding an intern, in particular...and leveraging tools like the Myers-Briggs Type Indicator or DISC assessments can help you navigate team...

... your gene of interest in a plasmid and get the vector incorporated into the germline. To begin this process...knockdown. Addgene also has a number of insect vectors to consider. An easy way to see what is available...Find Gal4 and UAS plasmids at Addgene Choosing a vector to generate a transgenic fly There are a few important...important points to consider when choosing the vector you’d like to use to generate your new fly line:... gene to be incorporated into the genome? Some vectors incorporate into the genome randomly, while others...would you like to select for your flies? Sure, the vector will have an antibiotic resistance gene to select...transgene. One such marker is the white gene, which restores eye color to fly lines where the endogenous white...

...bacteria that have taken up this vector, but it won’t tell you whether the vector contains the gRNA insert or...X-gal allows you to identify cells that took up the vector and distinguish between plasmids that contain the...functional 𝛽-galactosidase. Blue-white screens rely on vectors where the gene of interest is inserted into the..., you will see two bands: one representing your vector backbone and the other representing the insert....doesn’t contain the insert, you will just see the vector backbone band. Or, you can choose an enzyme that...specific to your insert or by using primers that are vector specific but amplify a potential insert. If you...plasmid does not contain the insert. If you’re using vector-specific primers, look for size differences. You...