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...gene at once. Therefore, applications that require simultaneous modification of all alleles of a gene,... recommends designing homology arms with the modification of interest, ordering them from a gene synthesis...with a homologous repair template. It is this application - the creation of sequence-specific knockins ...clones, making it unsuitable for most in vivo applications.  Third, even in the most optimized tissue-culture...tissue culture based approaches that require modification of only a single allele of a gene, such as the...efficiently deliver large repair templates for applications that require large gene fusions. The current...you’ve been able to check out some of the many applications of AAV in some of our other blog posts and have...

...beads through an assembly of biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP... (Figure 1). This enrichment streamlines the purification and minimizes sample loss while also supporting...scFvs for other targets or could be used for applications beyond cryo-EM.   Find plasmids for MagIC-Cryo-EM...interact with the air-water interface (AWI) before vitrification, they can be completely denaturated (left) or...chemical crosslinking or tilted-stage imaging. A truncated form of a tardigrade LEA, RvLEAMshort, protected...optimizing prime editing systems for specific applications! Delivering prime editors (PEs) via engineered...be delivered in a single package. In a recent publication, the Liu lab showed that this method of delivery...

...knowing that where you see fluorescence indicates your target’s location. Some other terms you might see associated...immunofluorescence? Generally speaking, IF is an application in which antibodies that have been joined to ...bind to a single primary, resulting in signal amplification. Another reason is cost effectiveness. Generating...distribute antibodies validate them in a variety of applications, including IF (or IHC or ICC), and will share...will share their findings either through their publications or as a review on a company website. It is important...Resources on the Addgene blog Learn about research applications of antibodies in Introduction to Antibodies ... for Me? Read about another common antibody application in The Basics of Western Blotting Resources ...

...pathways of DSBs, with a focus on repair for genome modification purposes. How does homology directed repair...specific mutations into the damaged DNA if these modifications exist in the template DNA. There are several... genome engineering world and made targeted modifications feasible and even easy. This targeted-break ...that can be harnessed by researchers for many applications. Your favorite Cas enzyme, a guide RNA to your...modified. In general, the insertion sites of the modification should be very close to the DSB, ideally less...oligodeoxynucleotides)) are commonly used for smaller modifications (~1-50 base pairs). Small edits require as little...Mouse Geneticist's practical guide to CRISPR applications. Singh P, et al. Genetics 114.169771; (2014)...

...neighboring fragments can be created via PCR amplification with primers that contain the appropriate homologous... tools that help you design primers for PCR amplification of fragments to incorporate such regions of ...assembly meets CRISPR Gibson can be adapted to more complicated cloning schemes, such as those where the vector...CRISPR technology and is described in a recent publication by the Lockey lab (Wang, et al. 2015). In this...aspects of the SLIC methods. First, it uses a dedicated 5’ exonuclease instead of using the exonuclease...DNA fragments contain these sites at the correct location and NOT in the middle of your insert. Gateway ...Gateway cloning utilizes λ integrase to catalyze directional cloning of DNA parts that are flanked by orthogonal...

...   Figure 3: Each publication featured in BioGRID ORCS has a dedicated summary page with PubMed...favorite gene was described in a CRISPR screen publication. You see this mentioned in the results section...manually curated and annotated from more than 45 publications, beginning with the seminal research articles...screen curated in ORCS is linked to the original publication via its PubMed identifier, which can also be ...a list of individual screens curated for the publication. Clicking on the screen name takes users to a... supplemental data tables available with the publication are also provided for the user. This approach...

...include: Cataloging a reagent’s performance as in this antibody ReFigure Analyzing replications as in this... ReFigure. Reading and exploration including replications and experiments resulting in “negative data”...small selection of data found in full-length publications. As early career researchers, we wondered how...reagent performs well, or you might publish a replication experiment that you conducted as part of setting...papers as they are not included in traditional publications. By submitting your data to a repository (Figshare...-registration, preprints, data, software, micropublications) in grant proposals. ReFigures are one such...

...advantage of a fusion protein is the efficient purification of an already immobilized target protein. The...immobilization method. One possibility is covalent modification of the purified target protein with the vitamin...positively-charged amino acids. Unfortunately, the location of biotin incorporation and to a lesser extent... ligand, especially if the modified amines are located near the ligand binding site. A selective approach...includes an AviTag and a 6X HisTag for affinity purification (see plasmid map and cartoon below). Including...erythropoietin molecules. The in vivo biotinylation and purification strategy outlined here provides a basis to determine...is barely soluble at RT. Track expression and purification of biotinylated protein on blots with Streptavidin-phosphatase...

...and attP recognition sites on the register and catalyzes their recombination, thereby changing the state...performed on one register at a time. If a particular application makes it difficult to isolate single registers...state read-out method may not be suitable for applications that require minimal cellular perturbation. ...The input plasmid (high copy, ColE1 origin of replication), contains large serine recombinase genes downstream...contains the register. Depending on the desired application, the inducible promoters we used to drive recombinase...register. RSM (See performance data from the indicated figures in our paper (1)) Input Plasmid Output...program to find the one that best suits your application. How efficiently do RSMs work? We tested a two-input...

...improve our shipment processes. After reading a publication describing how short term storage on dry ice ...medium, which is buffered and contains the pH indicator phenol red. We froze samples in a -80°C freezer...closed dry ice storage container for 48 hours (to replicate how our viruses are shipped), where they would...exposed to CO2/dry ice. All samples were tested in duplicate. What we found Not all tubes (or rather, not ... the samples were at the control pH of 7.4 as indicated by color (data not shown). What we did next: Venting... at -80°C. We then thawed the tubes after the indicated incubation periods, and measured the pH. We found...Clément N, Byrne BJ, Zolotukhin S. A simplified purification protocol for recombinant adeno-associated virus...

...2020.  “CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future.” Front. Oncol...with a dominant mutation in a gene that causes cataracts could be rescued by coinjection into zygotes of...the technology in different labs for different applications.” Li’s team noted that CRISPR/Cas9 has been ... explore its potential in mice with a dominant cataract disorder caused by a single copy of a gene known...with size limitations. In addition, non-viral applications are also possible through the use of nanoparticles....  “CRISPR Genome Editing Technology and its Application in Genetic Diseases: A Review.” Current pharmaceutical...

...including more physiologically-relevant systems, to catch all the important interactions. It’s also important... we found in our map have an impact on virus replication. While we and our collaborators are starting ... Nsp3 and Nsp16, and includes an additional catalytically dead version of the viral protease, Nsp5. Our... 2xStrep-tag for easy detection and affinity purification. All but one are in a pLVX vector background...coronavirus, one FedEx package at a time Since the publication of our paper, our group has been inundated with...sending plasmids to all who request them -- in locations all over the world, as far flung as the EU, Russia...Eckhardt and Melanie Brewer are on the Science Communications Team at QBI UCSF. They are particularly interested...

...protein for your application. Selecting the best fluorescent protein for a specific application can seem like...under conditions that best mimic the intended application. The three key properties for live cell imaging...are converted into emitted photons. These are indicated by the extinction coefficient (EC) and quantum...experimental set up. The practical brightness takes all application specific parameters into account, including the... picture of what can be expected in a 'real' application than the theoretical brightness. Consequently...brightness in a system closely mimicking the future application. In figure 2 we provide an example of the comparison...

...Addgene started distributing a small but growing catalog of ready-made AAV and Lentiviral preps. This new...directions. I would like to thank them for their time, dedication, advice and encouragement. Why virus? Why did...performs extensive quality control, generates  educational materials, and provides viral vector technical...answering questions on virus and helping provide educational resources on this collection. With their expertise... resource and providing fantastically useful educational content on viral vectors.  How Addgene’s Viral...about the new initiative and serving all the new educational content via the Addgene Blog and our expanding...the impression that it took a lot of work and dedication to do this well ... because it did. So finally...

...to the right indicates a higher signal, or upregulation, and a peak to the left indicates a lower signal...anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the percentage...case for you, switching to a contour plot should indicate where the two populations diverge. The smear ...place your gate based on the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing...Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes it easier to identify and study...addgene.org Addgene's Antibody Guide rAb Affinity Purification Protocol Video rAbs in the Addgene repository...

...editing applications.  Figure 4: Overview of CasMINI for genome engineering applications. Image ...stability in cells, and use it in a variety of applications. This set of new anti-FLAG frankebodies includes...to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling various targets... and anti-HA frankenbodies have a variety of applications, including live-cell imaging, single-molecule...members or students new to protein expression and purification. Figure 3: The Penn State Ladder run ...challenges for in vivo delivery and cell engineering applications. CasMINI, a new miniature CRISPR-Cas system ...

...enough to ensure the virus can replicate and spread. Instead, viruses replicate by hijacking the host’s system...low immunogenicity which makes them ideal for applications with whole organisms. Finally, viral delivery...delivery is typically straightforward across applications, while electroporation of a human, for example, isn...people in nature, extra safety measures, like replication incompetent viral particles, are required. We... (~4.5kb From basic science to clinical applications, viruses are true bench to bedside tools. Keep...our upcoming blogs on virus safety and viral applications to help you start (safely!) thinking about the...

... one half of naturally occurring restriction modification systems that prokaryotes use to protect themselves... has genes encoding one or a few restriction modification systems containing methyltransferases that add... phage for instance). Aside from restriction modification systems, DNA methylation also plays an integral...integral role in regulating genome replication, repairing mismatched basepairs or small indels that occur...methylases) are independent from the restriction modification systems, yet can still affect whether certain...specifically associated with any restriction modification systems, their sequences may overlap with restriction... Additional Resources Review of restriction modification systems NEB's Resource describing Dam and Dcm...

...competitions. After much learning, the focus of the application was changed, but Sample6 still leverages the ...fit for this set of technologies, effectively communicating the advantages embodied, and driving the enterprise...He explained that entrepreneurs must clearly communicate their idea at various levels of complexity and...backgrounds how amazing their idea is. While communication styles in academia and industry vary drastically...biggest challenge during this time was learning to communicate effectively to non-scientific audiences. "There...for the company; this will help avoid future complications and potential conflicts that waste money and...succeeding. Take home Starting a company requires dedication, passion, and around-the-clock work. Mike wraps...

...changes in expression of TRIM21-associated genes, indicating that TRIM21 overexpression does not aberrantly...light microscopy, comes from his recent Nature Communications article “SRpHi ratiometric pH biosensors for...250 nm), and studying endocytosis is further complicated by the reliance on pH-based biosensors used to...the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors to meet all of them. These...SRpHi Biosensors here. Richardson et al. Nat Communications. 2017. PubMed PMID: 28924139. FIRE-Cas9 ...system to study epigenetic modulation. While catalytically inactive cas9 (e.g. dCas9) has been fused to...plasmids can be found here. Braun et al. Nat Communications. 2017. PubMed PMID: 28916764. Plasmids for...