...assays such as mRNA data from transcriptomics; independent antibody strategies that compare the staining ...antibody that is directly conjugated to a detection substrate, such as a fluorophore, or a conjugated secondary...antibody validation.  The 5 pillars include genetic strategies that test expression in knock-out or knock-down...knock-down down tissues and cell lines; orthogonal strategies that compare antibody assay results with those...antibodies against the same target; tagged protein strategies that express a tagged version of the antigen ...antibody; and immunoprecipitation-mass spectrometry strategies that isolate an antibody’s immune complexes and...

... production and storage, we grow a little extra and transfer 100 µl of glycerol stock to each of 2 96-...through our deposit process, and then we carefully track samples through our lab using barcoded tubes and...such as special growth instructions (temperature, strain, media supplements), suggested sequencing primers...plasmid sequencing If you’re like us, you need a strategy for processing several sequencing reactions in... have different requirements for volume and concentration, and not all providers are equipped to perform...Your Plasmid Learn How Addgene Uses Barcodes to Track Samples Resources on the Addgene Website View ...

...milestones in the field. Research strategy The Research Strategy highlights i) the significance and ...weeks before the RFA due date in order for the administrative side of the grant to be established. Take time... information, selection criteria, and award administration information. Carefully read over all parts ...the best approach to the research challenge. Demonstrating expertise in the field and past successes boost...protocol, and offers alternative solutions to demonstrate thorough thought planning on the matter. The ...delegate funds to supplies, personnel, equipment, travel, etc. As a first time budget writer, you may find...

...later used by another team of researchers for neutralization assays, part of a study that examined the repertoire...effective sera from vaccinated individuals are at neutralizing the different SARS-CoV-2 spike variants. You...loop-mediated isothermal amplification after reverse transcription (RT-LAMP) and a colorimetric readout that can... 45 minutes. The team deposited the reverse transcriptase, protease, and Bst polymerase constructs from...toolkit include ones for expressing MMLV reverse transcriptase and Taq polymerase that can be used in a one-step...Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses 12:513. https://doi.org/10.3390...10.1126/science.abd0826 Tong P, Gautam A, Windsor I, Travers M, Chen Y, Garcia N, Whiteman MB, McKay LGA, Lelis...

...presentation as an opportunity to demonstrate technical and transferable skills that make you qualified ...position, I just had a phone interview. I also transitioned between different lab groups without having ...stories as the characteristic which the story demonstrates I possess.   Bullet points on resumes are like...spark a more natural conversation while also demonstrating that you did your homework before the interview... to highlight your prior accomplishments and demonstrate your science communication skills. Know your ...long! Mine lasted 4.5 hours, which was a lot of extraversion for me. Make sure to get enough sleep the night...

...should acquire during their PhD and postdoctoral training. Addgene executive director Joanne Kamens has ...scientific society in your field. My leadership training began with the NPA where I gained invaluable experience...issues. I applied and was successfully granted a travel award to attend the annual NPA meeting to learn...formats but I learnt to overcome the personal frustration and submit reports in the format requested which...leadership institute’ weekends would entail intense training to develop our leadership and management skills...to think about their career options, gaining transferable skills such as leadership skills can only assist...engaged once you are in a leadership role or in a track towards one.  Many thanks to our guest blogger Juliet...

...xCas9 for transcriptional regulation and base editing Hu et al. also engineered transcriptional activators...activating transcription. Sites containing NG, GAA, or GAT PAMs averaged 56-91% transcriptional activation...subunit ω. This construct is designed to activate transcription from the accessory plasmid (AP) encoding phage...could recognize a library PAM, it would induce transcription of gene III, permitting phage propagation and...pleasant surprise that selecting for one beneficial trait, PAM compatibility, also produced an enzyme with...

...variable regions that bind to specific antigens. In contrast, nanobodies are a single heavy domain fragment...involves using a commercial kit for in-vitro translation of the mRNA libraries. Each mRNA molecule in ... forming a ribosome complex consisting of the translated protein (sybody), ribosome, and mRNA. During ...enriched pool of sybody candidates are then reverse transcribed into cDNA fragments that are efficiently subcloned...response to the COVID-19 pandemic, the Seeger lab demonstrated they could use the protocol to rapidly identify...McLellan JS (2020) Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid...single domain antibodies for the conformational trapping of membrane proteins. eLife 7: . https://doi.org...

...One of the most powerful strategies to investigate a gene's function is to inactivate, or "knockout",...these techniques in mind when choosing a knockout strategy! Knockout plasmids Homologous recombination is... mechanism to accurately repair harmful double stranded breaks, in which nucleotide sequences are exchanged...endogenous promoter, you can use expression GFP to track cells participating in development or other physiopathological...very low, ranging from undetectable to 0.1%. In contrast, new genome editing techniques such as CRISPR ...created in 1989, less than thirty years ago. As traditional knockout and new CRISPR tools continue to be ...Cell Biol. 19: 1217. PubMed PMID: 19731224 PubMed Central PMCID: PMC2782548 Additional Resources on the Addgene...

... non-specific endonuclease (Cas9) and a single stranded guide RNA (gRNA). The ~20 nucleotide targeting...of wild-type Cas9 and a gRNA generates a double-strand break in the target DNA, which, when repaired through...heterogeneous population of gRNA-containing lentiviral transfer vectors, each targeting a specific gene within.... Pooled gRNAs are then cloned into lentiviral transfer vectors to create the CRISPR library. CRISPR libraries...most cases, CRISPR libraries are provided at a concentration that is too low for experimental use. Thus, ... first step is to amplify your library to a concentration that is sufficient to generate lentivirus. Be... you can skip the steps above! Cells are then transduced with lentivirus containing the CRISPR library...

...expressing neurons and GFP (green) to label neurons transduced by each serotype. Working at Addgene has ...provide an avenue to more easily solve these frustrating and often months-long quests to find the right... labeled high- and low-magnification images demonstrating vector performance) will be assigned citable...scientists to evaluate how small changes in vector administration can influence AAV expression properties.  By...providing DOIs, this information can be cited, tracked, and appropriately credited, broadening the knowledge...S., & Klein, R. L. (2018). More expansive gene transfer to the rat CNS: AAV PHP.EB vector dose–response...Kalwani, L., Sierra-Mercado, D., Robinson, J. O., Outram, S., Koenig, B. A., Pereira, S., McGuire, A. L....

...data for our DNA-based reagents. Not only do we extract as much information as possible from depositing...automating the bioinformatics steps needed to transform the raw sequencing data into a complete plasmid... to find a suitable software tool that could orchestrate the steps into a continuous pipeline. We also...appropriate place in our workflow without having to translate or convert programs to a uniform language.  The...concepts and vocabulary of Nextflow so that we could translate our pipeline steps into processes and then connect.... After this validation check, we repeated our strategy and copied a few lines of our existing script ...Bioinformatics AnalysesAAV Vector Quality Control: Going the Extra Mile with NGS...

... package stays in transit for a certain number of days and tracking average transit time for each courier...processes, and the distances the packages needed to travel meant the accessibility varied country to country...courier we contracted with so we can always offer the fastest shipping. However, there were issues that ...Protocols Agarose Gel Electrophoresis Bacterial Transformation How to Design a Primer Creating Bacterial Glycerol...Culture Collections Lentiviral Plasmids Tetracyline Inducible Plasmids Empty Backbones Bacterial ...

... but just as potent and specific? The two AAV strategies described above showed successful target modification...prime editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge...split AAVs. In general split AAVs break a large transgene in two pieces and package each piece into an individual... individual AAV. When a cell is transduced by both AAVs, the full length gene and/or protein is reconstituted...when at the ends of two proteins, mediates protein trans-splicing, similar to an intron in pre-mRNA splicing...DE, Brown MT, Deshmukh H, Kemp ML, Bao G (2015) Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes...2020) CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors. Cell 181...

...to be translated into Modern Greek to be easily accessible today. Roughly speaking, we translated our software...Addgenies, development includes everything from tracking plasmids from the moment a deposit kit is sent...Another advantage of Scrum is that it provides transparency to everyone involved about the status of each...details, step-by-step instructions on how to demonstrate the feature. For example, we recently reduced... Most of the time, updating these libraries is straight forward. However, there are times where a library...accelerating research and discovery. We work with transparency, gladly share our knowledge, and love to collaborate...

...the initial data-generating lab has efficiently trawled their own data? A distinct (perhaps similarly impoverished.... For example, mouse behavioral geneticists may track the motion of multiple animals on different occasions... available, but there remains concern about the extra work this may entail.  So if access to data is important...responsible for storage.  If datasets are too large to transfer to a journal repository, they are likely too large...repositories emerge that allow efficient searching and extraction of information. Looking a little further ahead...albeit unintentional, tendency to work against transparency. This opens science to charges of elitism and...opportunities for research as well as increasing transparency. Thank you to our guest blogger! Jim Woodgett...

...background on the different parameters that your gating strategy will be based upon. We’ll try to keep it short... promise! FSC and SSC: scattering To digitally extract your cells based on their size, the cytometer provides...background. Let’s dive into the praxis of gating strategies. As an example, we’ll use one of my previous ... from a C57BL/6 mouse and analyzed the tumor-infiltrating lymphocytes of that tumor. In Figure 3 you can...Figure 2), single cell events will show up in a straight line one the plot, while duplicates deviate from...Q1). If I wish to employ a more refined gating strategy, I can, of course, also draw multiple individual...Reading a Flow Plot Antibodies 101: Conventional vs. Spectral Flow Cytometry...

.... Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection by Alyssa Cecchetelli...Plasmid Cloning by Jennifer Tsang There are many strategies for finding your clones in an experiment. Read...Goedhart Guest blogger Joachim Goedhart shares some strategies for generating GFP fusion proteins. From considering...editing. Using a fusion between Cas9 and reverse transcriptase and a prime editing guide RNA, it can introduce...

...Training as a scientist in the academic system has many pluses. I delighted in my graduate school years...nature of academia often means that scientists-in-training rarely get the opportunity to learn about the ...science. I find it ironic that we do all of our training as scientists (5-12 years worth!) with academic... one of the best things you can do during your training is meet interesting people doing interesting things...connection that many people honor. Talking with strangers is usually challenging but does get easier with...you might spend a whole day having to talk with strangers. Be bold in asking all kinds of people to connect...

...after a holiday break filled with family, food, and travel, and you can’t remember where you put your flow...four white binders. Thirty minutes later you’re frustrated and no closer to starting your experiments. Get... deadline again. Organize you experiments, keep track of upcoming grant deadlines, and sync everything...Though electronic lab notebooks (ELNs) have gained traction with pharmaceutical companies, academic labs have...allow you to control who can edit the documents and track the revision history. Now you’ll be able to make... knowing the information is backed up and edit tracking will help you identify when others have added ...