...available in the adenovirus section below!   Fig. 2 - Viral vector components of AAV Adenovirus Adenoviruses...ITRs serve as a type of origin of replication to initiate replication of the dsDNA virus. Viral packaging...plasmid while the other plasmids with packaging or replication components are called helper plasmids. Below,... production, they can be helpful for certain applications and aid in ease of use. Our lentiviral guide...AAV are rep and cap. Rep is required for viral replication and also aids in infrequent genome integration...a helper plasmid and mediate efficient viral replication. More information on these viral genes is available...particles. E2A/E2B are responsible for viral replication, with E2B being the primary viral polymerase....

... a system closely mimicking the future application. In figure 2 we provide an example of the comparison...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...

...PubMed PMID: 8491198. PubMed Central PMCID: PMC413431. 2. Bukrinsky MI, et al. “Active nuclear import of human...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...

...acceptable variations in DR sequences. Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes...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 ...

...higher quenching of mScarlet emission. Figure 2: Ace-mScarlet is a fusion between the voltage-sensitive...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...

... and transfer 100 µl of glycerol stock to each of 2 96-well microplates (identical copies, see image above...digital notebook and make sure you note the freezer location of every plasmid in your collection. Use one of...access and use the materials they need, and avoid duplicating your effort. In addition, if you should choose... important step in ensuring quality, as it will catch any errors made during the cloning, sample handling... for more tips on sequence analysis, plasmid verification, and using NCBI BLAST. Tips for high-throughput...result against the expected sequence for quick verification, which can be done through the align tab of ... plasmid verification...

...JS, Legube G, Miller KM Genes Dev. 2015 Jan 15;29(2):197-211. DNA Cross-Bridging Shapes a Single Nucleus...transcription, or DNA repair, or replication, or any number of CRISPR applications and thought “OK, but how does...pages containing lists of plasmids from the indicated publications. You can request all of these plasmids ...below pull up lists of plasmids containing the indicated gene, and you can subscribe to these pages to ...

...divided by the off-target activity. Figure 2: Specificity is the measure of off-target activity...to generate a genomic alteration at a specific location, it must be accessible by Cas9. Our previous blog...off-target activity, which may be undesirable in many applications. At the time of our first blog post on choosing...CRISPR-Cas9 to increase its specificity." Nature communications 9.1 (2018): 3048. PubMed PMID: 30082838. PubMed...CRISPR-Cas12b for human genome editing." Nature communications 10.1 (2019): 212. PubMed PMID: 30670702. PubMed...

... those that also infect the liver.    Fig. 2: Summary of the generational evolution of the AAV ...of interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as a model organism...off-target mutations. These plasmids contain catalytically inactive dCas9 and microbial SSAPs (dCas9-SSAP...clonal multiplexing of cells in live imaging application. Each visual barcode consists of a fluorescent...transcription response element (TRE) or kinase translocation reporters (KTRs). Find visual barcode plasmids...

... high-priced commercial devices.     Fig. 2: Workflow for open software (left) vs. open hardware...convinced that most microscopy techniques can be replicated with commercially available consumer-grade components... tools has allowed the community to regularly replicate designs worldwide. Distributing hardware via globally...Hohlbein, et. al., 2022). A more open way of communicating in science brings stronger networking across...

...IgM, IgD, and IgG are deleted.     Figure 2: Isotype switching requires DNA recombination of the... Isotype controls are used in antibody-based applications including flow cytometry, immunohistochemistry...of these five isotypes, such as the number and location of disulfide bonds, the number of constant domains...

... would release them into the cytosol. Fig. 2: Endogenous sources of duplexed RNA DNA:RNA hybrids...generate dsRNA as part of their infection and/or replication process within the host. dsRNA viruses are inherently...generate dsRNA intermediates as part of the viral replication process. dsDNA viruses yield dsRNA through a ...

...state-of-the-art methods of sex and gender analysis and 2) provides case studies with concrete examples of how.... To remedy this, Shah et al. provide a table indicating the sex of the most commonly used cell lines ... work to be done! Researchers need to learn sophisticated methods of sex and gender analysis. Universities...

...biological applications. First, GMAP-compatible backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were...backbone, which expresses the gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene...engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand...number of tandemly expressed gRNAs respectively) and 2) the size of the deletion. Smaller deletions generally...FAIRE-seq, and CHIP-seq), genome-wide identification and quantification of enhancer activity have remained...compartments (cytosol, Addgene catalog #71245; mitochondria, Addgene catalog #71247) Rosella provides an ...CpG methylation by fusing catalytically inactive Cas9 (dCas9) with the catalytic domain from DNA methyltransferase...

...with abundance whenever possible.      Fig 2: Try to inversely pair fluorophore strength with protein...compensation.    Working with antibodies can get a bit complicated, but hopefully this helps you successfully select...

...about colony PCR in this blog post.   Figure 2: The steps of colony PCR.   Sanger sequencing Sanger... of the entire plasmid. Please note that NGS verification is more time-intensive than Sanger sequencing...

... the transcriptional level.     Fig. 2 Tetracycline on/off promoters and modes of action ...that! CRISPR interference (CRISPRi) targets a catalytically dead Cas9 fused to a transcriptional repressor...

...take care when using this method.      Fig 2:  A, The polyhistidine tag is a string of histidine...proteins within a system. This can be pretty complicated given that a single cell has tens of thousands...single protein? Oftentimes, they rely on protein purification. In this strategy a single protein can be isolated...this blog will focus on one of the most common, purification based on the polyhistidine tag. What is the ...purity (Janknecht 1991, Hochuli 1988). In this purification approach, the metal ions are immobilized on ... incorporation of a cleavage site and a post-purification cleavage step to remove the tag in order to ...2000).  One downside of polyhistidine tag-based purification, however, is the propensity for nonspecific ...

...inside the cell,” says Pedre Pérez. Figure 2: Micrographs of the biosensor expressed in the human... Haoyi Qiu He Tian Every two years, we seek applications for awards to early career scientists using ...virtual conference attendance. We also opened up applications to research technicians and undergraduate researchers...mercaptopyruvate sulfurtransferase (MPST) which catalyzes the desulfuration of 3-mercaptopyruvate and generates...the chromophore that are susceptible to redox modification, the MPST hydrosulfide can be transferred to... and CNO silencing. (D) Immunohistochemistry verification of the dual virus expression in CR neurons of...where she engineers genetically encoded voltage indicators. Tian developed a video-based high-throughput...

...plasmid (Option 1) or added using PCR-based amplification (Option 2). Advantages of Golden Gate cloning Golden... work around this is to "domesticate" your fragment: PCR-based amplification can be used to create silent...and exciting applications, this system is an incredibly powerful tool for cloning complicated constructs...Type IIS sites (such as BsaI or BbsI), that are located on the outside of the cleavage site. As a result...restriction sites that may not be amenable to "domestication", you might want to consider using an alternative...high-efficiency step. References: Sequential amplification of cloned DNA as tandem multimers using class-IIS...