...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. In 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...

... 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 ...

...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...

...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...

...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...

...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...

...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...

..., 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...

...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...

...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...