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...I led. It’s divided into the key takeaway themes and customer pain points we’ve identified. I can’t wait...software product management, I confess that I had no idea what software product management was either. Coming...

...developers of many of these probes, we decided to write this quick guide to help researchers choose the probe...supplied (Baloban et al., 2017).  It is important to consider effective brightness of NIR FPs in cells, in addition...binding of endogenous BV.  When imaging NIR FPs, consider using light sources that produce enough light ...NIR. Usually, mercury lamps are suboptimal, metal halide lamps are better, and xenon lamps are the best ...apparent cellular brightness than previously reported widely used dimeric iRFPs (Table 1). In general, the monomeric...O.S. et al. Single-domain near-infrared proteins provide a scaffold for antigen-dependent fluorescent nanobodies...

...but it isn’t just for lab specimens — there is evidence that lightning can also promote gene transfer ...While these plasmids can be dangerous, they also provide researchers with new means to deliver DNA and other...resistance to toxic substances like heavy metals or pesticides, enabling their hosts to survive where others... T. M., & Simonet, P. (2001). Laboratory-scale evidence for lightning-mediated gene transfer in soil. ...

...educational science videos is tough. Is the content accurate? Are we conveying complex ideas in the easiest... production standpoint, it wasn’t ideal for our Lab Protocol videos. It’s really nice to have a scientist...perfect height (Fig. 2). We recorded video using a simple, external video recorder and a mic clipped to her...pulled it off! Check out the PCR Primer video! Learn: Producing videos like this would prove to be difficult..., and as a video control center.   Cycle 2: Zooming Forward Design: We produced a video celebrating... cues. We had two options: Film outside at a distance, or film inside with one person in a room. I wouldn...protocol videos?   Figure 4: The remote directing setup, complete with wireless video receiver,...

...journal club. Sorting out the logistics Once you decide on the topic, there are a number of administrative...notice for the first meeting so that people can set aside time in their schedules and read the paper (PS: ... on single papers could be more important.  Any slides, presentations, recordings, and other materials... ensure that the journal club is useful for the widest audience, you should get feedback from those who...everyone to offer thoughts you may not have even considered. These questions should be tailored to the group...priority for the participants. Perhaps people feel confident in their own knowledge of the field. Perhaps interest... not actively participating, it may be time to consider ending the meeting. This is a common failing in...

...database of common features, and devising rules for identifying a feature even when the match is imperfect. SnapGene's...catalog every variant of a feature. Instead, we identified common variants, and then crafted a detection...reasonable rule is to require at least 96% sequence identity when detecting a reference feature. For a coding...match even when the DNA sequence has changed. Identifying plasmid control elements Coding sequence features...original literature, some of it decades old, to provide reliable feature annotations. Even with these extensive...that have different properties. To prevent misidentification, we augment the database with new fluorescent...protein variants. The future of DNA feature identification What’s next for common features? We plan to...

... it! Further Reading and Outside Resources http://www.businessinsider.com/how-to-get-the-most-out-of-job-fairs...your lab, right? Wrong! While it is clearly a good idea to be diligent in your research, remember that you...themselves, resources are often scarce and difficult to identify. So what specifically should you do? What follows... elaborated on later. As for what job fairs to consider, some of the best options are the Naturejobs Career...most relevant experience at the top. You may also consider visiting any of the employer’s career pages to... apply for the position. This post on Business Insider nicely summarizes good job fair practice. Career...and other online career resources Once you’ve identified places you’d like to work, get online and take...

...questions, data, and conclusions should be clearly identified and interpreted in less than a couple of minutes...Tips for designing your poster Check conference guidelines To begin, you should always follow conference...poster at all times, so other participants can identify you. You may also wish to make creative use of...three, or four column style, and in all cases the idea is the same; the reader will navigate the poster... to low, some of the poster will naturally sit outside of the reader’s field of view because of its length...to read. The Thompson Rivers University Library provides useful tips on how to craft this type of poster...doctoral student at Michigan State University. The idea is that the main research finding is written across...

...reverse transcriptase) together with a prime editing guide RNA (pegRNA). The pegRNA consists of a target-complementary...on the website or offline via GitHub. The model provides users with different designs of pegRNAs, ranked...and test them in their labs. Figure 2: Guidelines to predict pegRNA efficiencies with pridict.it...computational prediction models. In addition, we provide options for nicking gRNAs (used for PE3 and PE5...self-targeting screens such as PRIDICT/PRIDICT2.0 provide valuable insights into pegRNA editing efficiencies...Brinkman, E. K., Leemans, C., Vergara, X., van der Weide, R. H., Morris, B., van Schaik, T., Manzo, S. G....Addgene.org CRISPR: PRIME Edit Collection The CRISPR Guide Empty gRNA Expression Vectors...

...protein of interest, these tools provide an easier means to identify targets through fluorescent labeling...Lavis, L. D. (2022). Caveat fluorophore: an insiders' guide to small-molecule fluorescent labels. Nature...interiors to many inquisitive scientists. Seeing inside these specimens has offered illumination on biochemical...drug development (Grimm & Lavis, 2022). With the wide breadth of fluorescent tools that have been developed...will outline some fundamental characteristics to consider when selecting a dye or ligand to make your selection...protein, enabling the labeling of protein substrates/peptides and antibodies. Example applications of this include... is bound through green light emission. Key considerations in selecting fluorescent dyes/ligands Effects...

.... The video abstracts provide a brief introduction to the research and, after watching the video to determine...platform that provides links to the videos, keeping the copyright and control over the videos with the researcher...others. Improving science communication through video     Video abstracts about research, especially published...view count and videos published, YouTube reported a total of 865,995 views for the 926 video abstracts identified...attention for their work, a video abstract may be a useful tool for turning video views into online article...few years, I decided to look at what online tools people generally like to use to share ideas – with the... online video market ever since. The take home message of all of this appears to be that video is the ...

... CRISPR RNA Scaffolds." Adam has also created a video to help scientists visualize the concepts discussed...endonuclease activity coupled with a customizable single guide RNA (sgRNA) that bears complementarity to a 20bp...RNA (scRNA) will bind to a PAM-adjacent target nucleotide sequence then subsequently recruit a transcriptional...orthogonal dCas9 protein that will still recognize guide RNA but with a different dCas9 binding module, alternative... molecular microbiologist. Summary  The single guide RNA in the CRISPRi system can be repurposed to include... Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene ...

...have allowed scientists to easily perform genome-wide screens to effectively and efficiently investigate...libraries contain many sgRNAs per gene to increase confidence in the genetic screen. However, there are benefits...utilize only a few sgRNAs per gene. These libraries provide more information while using less resources and... the Broad Institute have developed human genome-wide CRISPRko (knock out), CRISPRi (interference), and... sgRNAs that were designed based on the rules identified in Doench et al., 2014 to predict sgRNA activity...scientists examined approximately 1,900 sgRNAs and identified sequence features and adjustments that improved...performance. The Root and Doench lab’s optimized genome-wide CRISPRko, CRISPRi, and CRISPRa libraries have been...

...gene function. In this final installment, I’ll provide a brief introduction as to how you can engineer...Addgene also has a number of insect vectors to consider. An easy way to see what is available is to browse...transgenic fly There are a few important points to consider when choosing the vector you’d like to use to ...pathogens and their hosts. It’s not always clear which side he roots for. You can try to convince him to use...

...deposition of the fertilized egg by its mother. Inside the pupal case, the larva will metamorphose into...light in coloration and can look very plump. On the side of their abdomen will be a dark spot. This is meconium...visible through their translucent bodies. This is the ideal stage to select for females because you can be certain...have an allele that causes the hairs on the dorsal side of its thorax to look like stubble (sb). For a better...search box in the upper right hand corner. They have video tutorials under the help tab like this one to get...pathogens and their hosts. It’s not always clear which side he roots for. You can try to convince him to use...

...things to consider: cost, time, equipment, and expected DNA concentration. It’s important to consider the advantages...DNA sample.  These absorbance measures give you an idea of the concentration of your DNA prep and whether...A280 ratio is used as an indicator of DNA purity. Ideally, this number should be between 1.8 and 2.0. The...containing a DNA intercalating dye (ex: ethidium bromide) and choosing a DNA ladder with known concentrations...

...FLEx Vectors Important Considerations When Using AAVs Three Key Considerations For Precise Neuronal Targeting...reproducibility of the data and can instill greater confidence when interpreting the outcome.    References...Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations...

...your machine!  Finally, many NanoDrop systems now provide full-spectrum analysis, rather than just the absorbances...Full-spectrum analysis allows the systems to better identify contaminants and to calculate corrected concentrations...critical interpretation of the results.  If you see evidence of contamination, you can try re-purifying the...these long enough?  There are, of course, more considerations and other approaches for measuring DNA quantity...use and check out some of the references we have provided below. And if you do have trouble with contamination...1ea8aec3bfa24543a28fcaea25986514.ashx. Koetsier G and Cantor E. A Practical Guide to Analyzing Nucleic Acid Concentration and Purity...

...the Dark Side." --Darth Vader in "Return of the Jedi" While Vader was referring to the evil side of a mystical...and rewarding. But, what if an FP fusion with a resident endoplasmic reticulum (ER) protein instead localizes...Previously, we have described a number of practical considerations that should be taken into account when deciding...must therefore determine your cloning strategy and decide whether you will create an N- or C-terminal fusion...needs. Equally important, before cloning you must consider whether and how well your FP will function in ...secreted into the extracellular milieu. Many of the resident proteins within these compartments undergo significant...post-translational modifications including glycosylation, disulfide bond formation, and proteolytic cleavage. FPs ...

.... Watch the following video to learn Daniel and Matthew's procedures. The video details how to transfect...Harvard Medical School. Addgene is proud to present a video reprint of the CRISPR article "Generation of Genomic...the Journal of Visualized Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs.... Biallelically disrupted cells can be easily identified. Even in the case of clones with monoallelic ...for biallelic deletion clones:    Like the video? The Orkin and Bauer lab's related paper describes... as their techniques for creating deletions, identifying these deletions, and finally characterization...

...just join one peptide tag to another peptide tag. Addgene: What is the advantage of peptide tags for building... locking two peptide tags together. SpyLigase overcomes limitations in the use of peptide tags, which ...assemblies? Howarth: Peptide tags can be very generic; you should be able to use the same peptide tag for all ...thanks for the T-shirt! References: SpyLigase peptide-peptide ligation polymerizes affibodies to enhance ...itself together, to produce irreversible protein-peptide interactions. In a study published in Proceedings...Developing a new tool Addgene: Where did you get the idea to develop SpyLigase?  Howarth: It came from our...the Streptococcus pyogenes protein domain made it ideal for this purpose? What led you to it? Howarth: Our...

...four of us would eat together every day, sharing ideas for the company, but also talking about sports, ...Addgene. We’re a small non-profit, so we can only provide free food on special occasions. However, employees...and make time for special projects? We borrowed an idea from the software folks and started Hack Days. During...plasmid protocols for our website. Free lunch is provided for participants during the Hack Days, and we’...Lunches” With more employees, Addgene has naturally divided into smaller functional teams. Our Executive Director...make time for free food, and these lunches would provide a friendly setting for people in different labs...Who knows what could come out of it? Perhaps new ideas or collaborations, or at least a shared sense that...

...practices and guidelines. It was wonderful to see the development of this practical guide by the workshop...networks, and specific neuronal populations to consider. To tease apart how different neurons impact a... discussions as a publicly accessible practical guide which was recently published in Frontiers in Neuroanatomy...experimental approach you choose, it’s essential to consider the technical limitations of each approach and...science.   Check out the precise neuronal targeting guide here! Additional resources on the Addgene blog ...retrograde AAVs Resources on Addgene.org Read our AAV guide Learn more about our viral service Find retrograde...

...living systems, and often do not fold properly once inside cells.  Developing the HA frankenbody To combat...genetically encoded antibody based probe that detects the widely used HA epitope in diverse cellular environments...epitopes (Ewert et al., 2004). This scFv hybrid provides the probe structure and stability from the scaffold...translated quickly by the ribosome. This feature provides HA frankenbody the ability to bind nascent HA ...a versatile tool that can be used to visualize a wide range of protein dynamics in vivo. In the future...Resources on Addgene.org Read our fluorescent protein guide Find plasmids for subcellular localization Find ...

... organization, contact me! I can provide a step-by-step, how-to guide to get one started. Advantages ...communication skills. The leader also provides materials such as pre-reads, videos or exercises to be completed...increases the diversity of input and perspectives Provides a good mechanism for accountability that makes...to talk about?” There are an infinite number of ideas on the internet, but start by downloading my handy...collected. The next blog in this series will have more ideas on what to do once you are in a mentoring relationship...Peer Mentoring relationships? Comment below with ideas from your own mentoring group or with any questions...

...one comprehensive, downloadable guide: the Addgene Science Career Guide. We also asked Addgenies what their...endeavor.  Break the process down into steps and set aside time during the week to tackle each one.  Get the...you’d like to see discussed on the Addgene blog, consider writing a guest post. Additional resources on...careers blog posts Read our transferable skills guide Resources on Addgene.org Find job listings at ...

...known as reprogramming [1]. The process can be divided into two stages: Dedifferentiation - Conversion...in vitro as part of the process. This opens up a wide range of possibilities in clinical situations. In...

...choice of junction sequences guided by experimentally-derived DNA Ligase fidelity data. Our work demonstrates...accuracy of assembly. The fidelity data can be applied to derive similar high-fidelity overhangs for any Golden...Reactions are often cycled between 37°C and 16°C to provide optimal reaction temps for both the restriction...appropriate levels of colony plating densities. Ligase fidelity and assembly efficiency Five fragment lacI/lacZ...redesigned for 12 and 24 fragments.  This redesign was guided by both advances in the re-engineering of the original...restriction enzyme and the completion of T4 DNA Ligase fidelity studies conducted by Potapov et al. at NEB (1,...Recently, NEB researchers profiled the comprehensive fidelity of cohesive end ligation by this enzyme for all...

...safe-targeting guides vs. non-targeting guides on screen analysis and hit calling. 5,644 non-targeting guides and...short guides had reduced toxicity compared to full-length guides. Reduced toxicity of short guides could...genes identified with short and long guides. Together these results suggests  that short guides have fewer...demonstrate safe-targeting guides as a potential control, and provide evidence that truncated gRNAs have...Genome-wide CRISPR/Cas9 screens are a high-throughput systematic approach for identifying genes involved...biological process. These screens provide an alternative to genome-wide RNAi screens, which although highly...questions about genome-wide CRISPR/Cas9 screen design Are non-targeting guides an appropriate control...

..., Moullier P, Ayuso E, Vandenberghe LH (2017) AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency...sequencing (NGS) QC platform that allows us to provide scientists with complete plasmid sequences. After... viral genome sequencing (VGS), to confirm the identity and serotype of our AAV preparations. We recently...an effective tool to confirm AAV vector genome identity. These studies used SMRT sequencing, TA-based ... that VGS can quickly and reliably confirm the identity of AAV genomes and detect contaminating AAV DNA...packaging cell line, helper and packaging plasmids. We decided to use this to our advantage by designing a Python...from multiple AAV lots can be run in parallel and provide serotype data in minutes and the scripts  can be...

...these benefits, iPSCs have increasingly become the ideal experimental system for research in regenerative...next post of this series, we’ll discuss the two widely used approaches of somatic cell conversion: conversion...

...Harvard University. In this second post in our quick guide to working with Drosophila, you’ll learn how to ... flies. Read the first post here. Once you’ve identified some fly stocks and other reagents of interest...pathogens and their hosts. It’s not always clear which side he roots for. You can try to convince him to use...

...This is the next step in high-throughput, genome-wide screening,” Konermann said. “If you want to activate...only do that screen with a system you are really confident about working for most genes and individual targeting...protein. SAM can be combined with a human genome-wide library to activate all 23,430 known coding isoforms... we could use information from the structure to guide and improve activator design,” she said. “Before...exposed RNA loops, which Konermann recognized as an ideal spot to fuse the activators instead of at the protein...consistency and validation with >80% effectively enriched guides per gene hit, and 100% validation of the top 10...genes of interest. They also “showed that different guide RNAs have different abilities to turn genes on and...

...enzymes." Science 362.6416 (2018): 839-842. PubMed ID PMID: 30337455. Additional resources on the Addgene...systems were merely adaptive immune systems that provide bacteria protection against infectious agents. ...predicted that many others are still unknown. Identifying Cas14 variants In this quest to discover new ...that improved specificity of Cas14 enables high-fidelity SNP genotyping. The development of  Cas14-DETECTR...Find the Cas14 plasmids here Checkout our CRISPR guide page Browse the CRISPR collection ...

... careers outside of academia during my graduate training. PhDs have many roles to play inside, on the ...away screaming, but instead can involve walking alongside it and giving it a high five. While screaming ...the outskirts, and entirely outside of academia. These roles can include positions, like mine, that help...

...in the apo state (PDB ID 4CMP, top) compared to the guide RNA-bound state (PDB ID 4ZT0, bottom), seen from...structure (left, PDB ID 4ZT0) with Cas9:RNA:DNA electron microscopy structure (right, PDB ID 7S4X), viewed from...bound to guide RNA (PDB ID 4ZT0), shown opaque (left) and transparent (right). The RNA guide sequence ...Figure 5: S. pyogenes Cas9 bound to guide RNA and target DNA (PDB ID 7S4X). Residues R1333 and R1335 from...structure of S. pyogenes Cas9 bound to guide RNA and target DNA (PDB ID 7S4X), shown opaque (left) and transparent...Figure 7: S. pyogenes Cas9 bound to guide RNA and target DNA (PDB ID 7S4X). The end of the DNA:RNA heteroduplex...structure of S. pyogenes Cas9 in the apo state (PDB ID 4CMP) with domains colored as in Figure 1. HNH, red...

...with the Plasmid Name, Addgene Plasmid ID #, and the PubMed ID # (PMID) for the article that has used ...it has been validated in.  If a plasmid's Addgene ID # has been referenced in other publications, you'...used your plasmid of interest, which may help you identify relevant protocols and experimental data Find...

...we request that you cite it using both its Addgene ID number as well as the reference for its original ...cite a plasmid from Addgene using its unique Addgene ID number, your publication will be listed on our website... You can search for plasmids by name, PI, Addgene ID number, or keyword. Although an initial search might...plasmids from Addgene right on our plasmid pages and provide a citation that you can put straight into your ...search results using the filters found on the right side of the search page. A great breakdown of our search...search functionality can be found in the following video: Please remember that the accuracy and reliability... Blog Post on Our Citations Feature Browse More Inside Addgene Posts Additional Resources on Addgene....

...were able to include specific plasmid ID numbers. If you know the ID number for a plasmid, your straightforward...by our own CSO. But what if you don’t have plasmid ID numbers, or you aren’t sure if any plasmids from ... associated plasmids through the article’s PubMed ID (PMID) ...or DOI Or get some keywords close enough...more. Selecting crispr in Guides will return links including our CRISPR guide.  For a closer look at this...search. These pages provide helpful information about the subject, link to related guides and protocols, and...delivering a calcium biosensor using AAV, you might decide you’d really prefer to order ready-to-use virus...search will start from a publication. You may have an idea for a plasmid that would be useful in an experiment...

...entering the data. This step associates an Addgene ID with your plasmids, and adding all the plasmids at...likelihood that the plasmids are given sequential IDs.   Name – we encourage the use of descriptive plasmid...include the total size of the insert and the GenBank ID associated with your insert. Pro Tip! If the correct...and the vector type. Pro Tip! Reference the Addgene ID in the "Backbone Manufacturer" box for backbones ... Confirmation email from us with a unique Deposit ID. Addgene will send you a package containing instructions...scientists worldwide. What's more, depositing is free! We've written this post as a step-by-step guide to the...contacting [email protected]. These deposits are considered large deposits and are exceptions to our standard...