...Susanna Bachle, Addgene the nonprofit plasmid repository 49:45 0 1:18:45 - Reagent Development - Steven...Alex Tucker (Ginkgo Bioworks) Pamela Hines (Senior Editor at Science) Edward J. Hall (Professor of Philosophy...sequenced and quality controlled plasmids and viral vectors. When working with a plasmid from Addgene, you ...negative” data We all want to be able to tell a good story when presenting scientific findings. This makes ...of information and, from a SciComm perspective, stories with links of causation from initial hypothesis...validation are easier to understand. The need for great stories has, however, made it difficult to publish so-called...original hypotheses. Looking for novelty in our stories, we are additionally averse to producing papers...

...industry, non-profit, and government sectors.  Within these sectors, you could start your own company, ...based on any or all the contents of this post.  An attorney should be contacted for advice on specific legal... as law, medical/technical writing, clinical, regulatory, product development, business development, consulting...career paths in academia as well as the non-profit sector are transferrable to industry such as technology...greater scientific field, potential customers, investors, or other relevant entities or individuals. A ...peer reviewer for journals, acting as a journal editor) of the work of those in your field. Original contributions...papers that you should start contacting journal editors to request to be a reviewer. Proactivity is very...

...time: Functional genomics research in an introductory laboratory course. CBE-Life Sci. Educ. 17, ar1. (2018...experiences to undergraduate students (1, 2), but educators face significant challenges in designing suitable...could provide the basis for student projects in introductory labs. Genome projects use computational methods...research questions. The idea is straightforward. Instructors first select a multi-gene pathway to study and...yeasts are thought to have diverged from a common ancestor ~350 million years ago. Over time, the two genomes...species into the pYES2.1/V5-His yeast overexpression vector. This plasmid contains origins of replication and...research skills (8). A complete set of student and instructor resources is available at our Investigations ...

...proteins for use as FLIM-FRET acceptors  Listen to the FLIM-FRET acceptor podcast segment Article contributed...FLIM-FRET acceptors here. See Addgene's FRET reference page for many other common FRET vectors. Murakoshi...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...termied soCoChR, by fusing a fragment of the kainate receptor subunit to the C-terminus of the protein. Unlike...soCoChR-expressing cells. Finally, using synaptic receptor blockers to distinguish real and artifactual postsynaptic...placesTagRFP-T and Dronpa in close proximity. By monitoring changes in TagRFP-T fluorescence intensity at...Murakoshi lab has developed two new FLIM-FRET acceptors, ShadowG, a novel dark green fluorescent protein...

...for drafting my stories. In total, I prepare 5-6 stories for an interview, since one story usually has more...Instead, I preferred to prepare stories to answer interview questions. Story telling shares many of the same...of the theme of my interview stories as the characteristic which the story demonstrates I possess.   Bullet...helpful for my interviews in industry. Prepare stories to tell in your interview The STAR interview method...answer: a setting, a conflict, and a resolution. Stories usually have a theme too, which is something not...of questions. Writing a rough outline of these stories helped me remember them for the interview.  Prepare...

...including a handful of plasmids that have been in the repository for many years, has now grown into a collection... spike protein to bind ACE2, the host cellular receptor. To more easily study the spike variants, scientists...Choe’s lab identified that ACE2 is the functional receptor for SARS-CoV. The lab also found that cathepsins...have an important role in viral entry, which are inhibitors of SARS-CoV-2 entry and become upregulated during...pandemic, the Krogan lab at UCSF generated lentiviral vectors expressing SARS-CoV-2 open reading frames and identified...proteins which all but one are cloned into pLVX vectors (for lentiviral packaging) and contain an IRES-...above, some of these plasmids have been in the repository for many years while many were developed during...

...primers that target the common features of viral vector backbones; once validated these universal primers...higher than for functional assays depending on the vector backbone. When using qPCR based methods for viral...plasmid which can over-inflate results. A packing vector deficient in reverse transcriptase will produce...the number of target cells that are positive for vector encoded transgene expression following transduction... prep. This method is particularly useful when vectors carrying fluorescent markers are used as it eliminates...significantly higher than the same prep that has been stored in the freezer. If you plan to freeze a batch of...titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009): 495-505. PubMed PMID...

...We’ve dug into the data from our repository to find our most requested plasmid in 2015. In line with ...that the plasmid most requested from the Addgene repository in 2015 was... lentiCRISPR V2   LentiCRISPR...fantastic research tool has been requested from the repository nearly 1,000 times in 2015! If you haven’t already...suggests, lentiCRISPR v2 is a lentiviral transfer vector used to deliver both Cas9 and your gRNA of interest...lentiviral titer as compared to previous lentiviral vectors. It can be used both to knock-out single genes ...is available from Addgene both as a single empty vector and in pooled libraries containing thousands of... has already been used for includes work probing mTOR signaling in cancer cells, deciphering the mechanism...

...recently we have also been using Addgene CRISPR vectors to generate cell lines with knockouts of specific...specific genes. Many years ago, people in the laboratory became frustrated with using glass cloning rings ...sterile truncated pipet tips. Indeed, an experienced operator can pick 48 clones from a 10 cm dish in 15- 20...medium containing serum in each well. A 200 ul pipettor is set to 200 ul, wiped down with 70% ethanol,...individually punched out, gently sucked up into the pipettor, placed into wells, and partially dislodged from...YouTube video. Before returning the plate to the incubator, additional growth medium is added to about halfway.... We highly recommend the use of an electronic pipettor with variable spacing (such as the Viaflo from...

...variant can then be fused to effector domains such as transcriptional activators or repressors. These fusion...cut either strand of DNA. Traditional Cas9 regulators In addition to its utility in making targeted...to DNA, Cas9 can be reprogrammed to serve as a regulator of gene expression. Its catalytic domains can ...hairpin, that is capable of recruiting additional effector domains (4). Taken together, these minor gRNA ... generate a potent Cas9-based transcriptional regulator without making any functional changes to Cas9....adapter capable of recruiting a transcriptional activator, robust regulation of gene expression is observed...the truncated gRNA recruits a transcriptional activator to the Cas9-gRNA complex. When this complex is...

... and other viral vector tools in the years to come. We’ll make many more viral vector tools and resources... Service Page Browse Additional Viral Vectors Find Viral Vector Protocols ... retrograde (the most popular form), this viral vector is used to deliver DNA encoding Cre recombinase...be sure to check back often! To find more viral vectors available through Addgene, be sure to check out...out our viral service page and browse our viral vector blog posts for many more tips and ideas on how to...thanks to Hongkui Zeng’s lab for providing this AAV vector! Additional Resources AAV Titers: Where do they...for Gene Expression in Mammals Subscribe to Viral Vector Blog Posts Additional Resources on Addgene.org...

...the Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed...activation using a fusion to a transcriptional activator such as VP64 (Gilbert et al., 2013), or repression...region to inhibit association of transcriptional activators (Qi et al., 2013). However, the fusion of dCas9...specific genomic region In 2013, the Hodaka Fujii Laboratory first described a method to purify a specific...interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an antibody targeting...lab is CRISPR affinity purification in situ of regulatory elements (CAPTURE) (Liu et al., 2017). This system...miR483) that binds to the promoter of the growth factor IGF2 open reading frame to relax imprinting (Zhang...

...catalytic protein and its cofactor VP35. VP30 is a transcription enhancement factor. When used to study minigenomes...Addgene.org Browse Our Viral Vector Plasmids Read Our Lentiviral Vector Guide Read Our AAV Guide  ...producing infectious virus (similar to lentiviral vectors) because they do not encode all of the necessary...changing the minigenome cis-acting sequences and monitoring effects on genome replication and transcription...Microbiology, National Emerging Infectious Disease Laboratories.  She is interested in genome replication and...Karl-Klaus Conzelmann. "Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is...on the Addgene Blog Read Other Posts on Viral Vectors Check out Our Scientist's Guide to Ebola Contribute...

...with GeneWeld vectors GeneWeld from Jeffrey Essner’s lab uses CRISPR/Cas9 and a donor vector to create knock-ins... tools for your research! This time: GeneWeld vectors to create knock-ins CasX Drug inducible CRISPR/...proteins, fusion proteins, or transcriptional regulators. In this system, Cas9 generates a double-stranded...cargo (ex: fluorescent protein gene) on the donor vector. Therefore, targeting requires two sgRNAS: one ...site and a universal sgRNA that targets the donor vector sequence. Homology arms that flank the donor plasmid... Read the paper in bioRxiv Find the GeneWeld vector kit CasX, a small Cas protein, is active in E....editing by fusing Cas9 to a mutated human estrogen receptor. This protein fusion stays in the cytoplasm, but...

...populations using combinatorial viral approaches. Current protocols in neuroscience / editorial board, Jacqueline...and for good reason. For the first time in the history of neuroscience research we have a technology that...spent two months in the lab of Antonello Bonci, Director of the National Institutes of Drug Abuse Intramural... electrophysiology data too but that’s what collaborators are for). For example, if you study neural pathways...his optogenetic constructs available through the vector cores of various universities as well as Addgene...transfer agreement, and list of vendors (such as the vector core at the University of North Carolina) that ... Conduct a time course experiment in which you monitor expression at various time points (e.g. every 1...

...recently was invited to attend the first Principal Investigator meeting of the NSF EDGE (Enabling Discovery ...Discovering ways to culture, propagate, transform, store, and share all of these new organisms continues ...continues to be challenging. Organism specific viral vector systems would also be beneficial for many systems....unavailable.” As stated by one of the NSF program directors, this program should “enable the ability to study...parenting strategies and make important ecological indicators since species live in water and on land. Current...that tastes yucky (and is poisonous) for bird predators - birds eat a caterpillar once, vomit, and never...humans learn speech. Finches must learn early, use auditory feedback to learn, babble as baby birds, and have...

...was contributed by Erik Snapp, the Director of Student and Postdoctoral Programs at the Janelia Research...Structural Biology, and now serves as the Director of Student and Postdoctoral Programs at the Janelia Research... payoff for each aim. Learn to communicate your story with only the most salient details. Make the most...your research problem. Creating a coherent unified story and model can help make your ideas easier to understand...Practice presenting your chalk talk Perfect your story and delivery. Design simple figures and diagrams...track. Furthermore, practice getting back to your story. Committee questions can derail your talk. You need...from Oregon Health Sciences University, did his postdoctoral fellowship with Jennifer Lippincott-Schwartz...

...software. The Inkscape website has many tutorials and Lynda has a tutorial as well, which you may have access...professional-grade design software, like Adobe Illustrator, and I didn’t want to pay for these programs ... Vectr is like a pared down version of Adobe Illustrator. There’s both a web and a desktop version of ...layers, which let you lock and hide individual vectors (shapes defined by 2D points connected by lines...Inkscape Inkscape is the most similar to Adobe Illustrator out of the three options for drawing your own...and editing shapes and like Vectr, Inkscape uses vector graphics. There’s a steep learning curve for Inkscape...20,000 icons is designed by professional medical illustrators with input from life scientists. It’s easy to...

...would love to tell such a dramatic story, but it wouldn’t be my story. I love academics. If I didn’t, it... took on TA positions, tutored fifth graders, made a blog and worked as editor-in-chief for the Science...there was even a time when I was too nervous to ask store clerks for help at the mall. In undergrad I was ...thought that I would be best poised to share the stories and difficulties behind great discoveries by tackling...largely my work developing myself as a science communicator that got me here! Thus far being an Outreach... mission to make research easier. I’m hoping my story demonstrates that transitioning from academia doesn...Management for Scienitsts from Addgene Executive Director Joanne Kamens ...

...It’s nearing the end of #DepositorWeek at Addgene and we’d like to show our gratitude on the blog by ...highlighting some of the great posts written by Addgene depositors. Below you’ll find brief summaries of several...several excellent guest posts from our depositors. Interested in writing your own post for the Addgene Blog...Tools In this post, Jessica Polka, formerly a postdoctoral researcher in the Silver Lab at Harvard University...them. CRISPR Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening CombiGEM is a technology that... organism. In this post, Chris Richardson, a postdoctoral researcher in Jacob Corn’s lab, describes how...Albert Cheng and Mark Wanner from the Jackson Laboratory for Genomic Medicine described Casilio, a system...