...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...

...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...

...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...

...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...

...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...

...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...

... 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...

...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...

...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...

...enough space! There’s enough space here at our new location to grow. We are hiring again and getting ready...to keep up with the demand with excellence and dedication. We don’t have worry or wonder if providing...research, hosting conferences, and are expanding our catalog of materials to better serve the community. We ...projects allows us to support more new projects. Diversification is happening. The Addgene team will have some...we might do. I am grateful every day for their dedication, flexibility, innovative thinking, and openness...Addgenies support a high collaboration, high communication, high efficiency, high quality, low meeting,...about the Addgenies. I never worry about their dedication, excellent intentions, or incredible drive to...

...voltage indicator Article contributed by Shreya Vedantam Genetically encoded voltage indicators (GEVIs)...fluorescent optics. Existing red fluorescent voltage indicators, however, fall short compared to GFP-based sensors...linker between them. The Ace-mScarlet voltage indicator works like many FRET-opsin GEVIs. With an increase...channelrhodopsins. Find the Ace-mScarlet voltage indicator plasmids at Addgene! Beck et al., Sci Report 2019...increasingly been recognized for its biotechnological applications including conversion of CO2 into water and electrical...the N-terminus of Cre (CreN) are fused to the truncated Phytochrome B (PhyBΔ) and the active protein binding...

...re used in a number of research and clinical applications. Most of the time, when someone speaks about...deeper penetration into a tissue, useful for applications like IHC, or be able to access an epitope folded...that would size-exclude an antibody. In imaging applications, smaller affinity reagents means higher resolution...antibody’s ‘Y’. They are often used in clinical applications. Fab fragments can be created in lab in situations...Shahied, 2004). They are often used for clinical applications, including imaging.  Diabodies are ~60 kDa proteins...antibodies are still an excellent option for many applications, and frankly, if ain't broke, don't fix it!  ...flexible in your approach to affinity reagent applications. Whatever affinity reagents you use, one thing...

...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...

...summarized in a recent Nature Medicine paper. In the publication, Bhang and coworkers used the ClonTracer library...propagated it, separated it into roughly identical replicate groups, and treated these groups with the same...below): 1. Resistant cells among the separate replicates would contain the same labels - i.e. they would...subpopulation 2. Resistant cells among the separate replicates would have different labels - i.e. they would... Looking at barcode abundances among multiple replicate populations, they found that similar barcodes ...populations were shared by at least one other replicate; prior to treatment, there are small (previously...such that initial resistant populations can be eradicated up-front quickly. This approach is in contrast...