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...available. Table 1 compares some general advantages and disadvantages of each approach. Table 1: Properties...nighttime displays, these insects have contributed an important tool to the scientific community: luciferases,...levels and improved signal sensitivity. Another advantage: luciferase reporters aren’t susceptible to photobleaching...Japanese firefly (Luciola cruciata), and more. These variant proteins offer different kinetics, substrate requirements...luminescent substrate. This approach preserves the advantages of luminescent assays (low background, no photobleaching... that produce different colors or have other advantageous properties. This approach can improve live-cell...measurements of luminescence from live cells. Luciferase variants with incompatible substrates can also allow multiplexing...

...macromolecule density is equal to the buoyant force of the solution. Contaminants of lower densities are captured...Iodixanol Gradient Ultracentrifugation First, you’ll want to identify the reader’s goal. Why are they reading...What is the goal, who is my audience, and what do I want to be different for them when I’m done? What are...wait!  Done? Excellent! We asked our workshop participants to do the same thing, and even though only about...between layers are muddled due to the passage of supernatant through each layer. The right panel illustrates...before and after ultracentrifugation. The viral supernatant migrates through increasingly dense layers until..., and we’d love to hear yours as well.  More importantly, you can use these four steps for any type of...

...comparison of different enzymes. CasPEDIA is meant to be a constantly updating database which serves as a resource... Links to relevant resources The homepage displays a graphic describing CasIDs and relevant properties...field has exploded with dozens of Cas enzymes and variants available. Without a comprehensive resource, it... What are the features? CasPEDIA has collected relevant information on all the Cas enzymes you know and... know for viral packaging limitations. Maybe you want to read literature on the less common applications...experiment. Perhaps you are a structure junky who just wants to learn about Cas domains. CasPEDIA either directly...CRISPR–Cas systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https...

...function in terms of its regions. But equally important is the way the brain connects across these regions...is connected to itself across these regions is important for understanding how the brain functions. The...neurons that receive information from a region (anterograde tracer, Fig. 1B). Tracers are typically delivered...the soma (neuron cell body) toward the axon (anterograde tracer) or upstream from the axon terminals to...University of California, Berkeley engineered an AAV variant that efficiently infects neurons, directs high-...defined population of neurons. For example, say I wanted to access all the neurons that signaled from the... spinal cord (Wang, 2018, Metcalfe, 2022) and substantia nigra (Tervo, 2016). Discrepancies in transduction...

...cells you want to deliver to, and how you’ll be measuring your experimental output. You’ll also want to keep...serotypes can be transported retrogradely and/or anterogradely along the axon. Others, like AAV1 and AAV9, ...going to be working in several different systems or want to transduce a large number of different cells. ...et al., 2014; Nieuwenhuis et al., 2021). It is important to note that this expression pattern can be affected...however, increases with the size of the region you want to cover - and as your titer changes, so may your... is one that optimizes gene expression where you want it and minimizes expression where you don’t. The...Nonhuman Primates after Intramuscular Injection of Recombinant Adeno-Associated Virus. Journal of Virology, ...

...the hM4Di, we used primary antibodies against mCherry, and secondary antibodies coupled to nanogold particles...Fluorescent proteins can also be identified using antibodies and immunohistochemistry techniques. Thus, these...transgenes of interest. Furthermore, fluorophore variants (for example green and red fluorescent proteins...Replacing the mCherry with the HA tag resulted in abundant expression of hM4Di in plasma membranes in both...results indicate that the tag protein may have an important influence on the expression of the transgene of...plasma membrane. Note that the use of the HA tag significantly improves the transport of hM4Di to the plasma...studies suggest that non-fluorescent tags may be advantageous over fluorescent proteins when the fluorescence...

... CRISPR’s therapeutic potential in mice with a dominant cataract disorder caused by a single copy of a...co-injection of Cas9 and a single-guide RNA targeting the mutant allele. When all was said and done, the researchers...therapies that are currently approved or have made significant progress in clinical trials.     Figure...almost all patients experiencing sustained and substantial relief from both disorders. As of publication...effectiveness, and delivery. Patient safety is important for allogenic therapies to prevent a graft vs....vs. host response. Off-target effects are also important safety considerations for all CRISPR therapies...Thus far, several targeted therapies have taken advantage of what our current delivery technologies are ...

...delivered using viral vectors! One of the most significant advantages of viral delivery is the potential for ...Calcium is arguably one of the most important metal ions in biology (sodium and potassium are up there...start by asking ‘What is the biological phenomena I want to observe?’ This information, along with a little...your experiment could be subtle or dramatic. You want to use a calcium sensor that is most sensitive to... than in the cytosol. Thus, for the ER you would want a low affinity calcium biosensor, so that the (1... with an extra apoCaM-binding motif to prevent aberrant binding of the biosensor to endogenous calmodulin...CEPIA) or through FRET sensors, can reduce these unwanted variables. Twitch is a FRET-based calcium reporter...

...with a deletion-mutant rabies virus. Retrograde neuronal tracing with a deletion-mutant rabies virus. Pubmed...that the body’s architecture and plumbing are important. We started with learning the head is connected...neural connectivity (Nassi et al. 2015). The recombinant viruses typically used in neuroscience research...transmitted transsynaptically). This is because the recombinant forms of these viruses typically used in research...monosynaptically from these cells. These modifications take advantage of the fact that RABV is an enveloped virus. In... (Watabe-Uchida et al., 2012). They could also quantify the strength of the inputs by counting the monosynaptic...characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study. J Gen Virol. ...

...Taking advantage of Golden Gate cloning, the Haynes lab’s Protocols.io protocol describes an elegant system...also be applied to other fluorophore/antibody pairs. Because the anti-GFP nanobody cross-reacts with BFP...length can and should be varied as it can have important implications for the fusion protein effectivity...GFP on the surface and “receiver” cells express an anti-GFP nanobody on the cell surface. When the cells...both spatiotemporal protein degradation and quantification of this depletion using imaging-based measurements... the degradation of the AID-tagged protein. To quantify protein degradation the Ward lab used fluorescent...levels of dCas9 expression and contain different antibiotic resistance markers. New CRISPR pooled library...

... as a successful applicant to the Addgene, Seeding Labs Plasmid Grant. For this grant, we’re working with...about the Addgene, Seeding Labs Plasmid Grant Read about Plasmid Grant Recipient, Gladys Kaba Resources on...their research. Kwabena came across the plasmid grant in Seeding Lab’s Newsletter and recently visited...Kwabena applied for the Addgene, Seeding Labs plasmid grant. 7:12 - 7:11 : Kwabena's work in Christopher E. ... recipient of the Addgene, Seeding Labs Plasmid grant and is working on a variety of projects in Ghana...

...Technique Sequence Information Detection Limit Advantages Disadvantages Mismatch Cleavage Assay Not Provided... Technique Detection Limit Application Advantages Disadvantages GUIDE-Seq 0.1% Cell-based  Searches ... the University of Cambridge. One of the most important steps in the CRISPR experimental process is validating...silico prediction. These techniques differ in important ways, but can also complement one another by providing... amplicon sequencing (Hendel et al. 2015), a significant difference as CRISPR experiments move closer ...networks in biological systems. NGS will also play significantly into the realm of clinical therapeutic development...sequencing: a comparison of methodologies for KRAS variant detection." Biotechniques 53.3 (2012): 182-188....

...guest bloggers, Jae Lee and Pantelis Tsoulfas. Jae K. Lee is an assistant Professor at the University ...This post was contributed by Jae Lee and Pantelis Tsoulfas of the Department of Neurological Surgery ...arrows point the direction of the specimen, A= anterior, P= posterior, D= dorsal, V= ventral Developments... milliseconds to acquire one image, thereby significantly reducing the time it takes to image through ...Similar to light microscopy, there have been significant advances in tissue clearing techniques during...which is a hydrogel-based method that also allows antibody penetration for immunohistochemical labeling in...Medicine. Find plasmids from the Lee Lab here. Pantelis Tsoulfas is an Associate Professor at the University...

... using a dCas9 variant as well. Furthermore, they provide a high-fidelity (HF) variant of the BE3 that...Addgene depositor Luciano Marraffini’s lab took advantage of this lethality to design the first synthetic...the original pCas/pTargetF but has many other advantages including shorter plasmid curing time. pCRISPomyces... a mammalian cell line (Qi et al., 2013). Two important features of this early system are its regulatable...bacteria. The authors were able to engineer a dCas9 variant fused to bacterial enhancer binding protein required...could be genes, specific polymorphisms, including antibiotic resistance genes. This approach successfully ...Streptomyces Some of the most industrially and clinically relevant bacterial species reside in the genus Streptomyces...

...for AAV Quantitation by Kate Harten DeMaio We made the switch to droplet digital PCR to quantify AAV titers...development virtually. 6. Quantifying DNA? Here are Five DNA Quantification Methods to Consider by Jennifer... dominates DNA quantification discussions, but what about the other methods to quantify DNA? Here we cover...Methods Based on CRISPR/Cas by Shravanti Suresh Guest blogger Shravanti Suresh who studies CRISPR at Iowa...

...challenge when I started working with AAV vectors. I wanted to use an AAV vector to express ChR2(H134R) in ...experiments didn’t provide any information on other important factors such as what is the best promoter to use...virus to adjacent areas impacted the experiments we wanted to perform. It required a great deal of time and...Addgene Data Hub, 76% of respondents indicated they wanted to see more negative data (Figure 2.) At the same...help each other by sharing their data. So take advantage of it and get your data - negative AND positive...T. R. (2015). Stability and compatibility of recombinant adeno-associated virus under conditions commonly...Lutomski, C., Jarrold, M. F., Boulis, N. M., & Donsante, A. (2019). Lot-to-Lot Variation in Adeno-Associated...

...Regardless of the type of DNA you want to purify or your motivation of wanting to go kit-less, there’s a method...DNA without a kit. DNA purification kits have advantages: they are convenient and provide uniform, consistent...Cellulose nucleic acid purification Starting material: plant, microbe, blood, or mammalian cells or tissue Product...least entraps nucleic acids. The Botella lab took advantage of this property and developed a protocol that...or even paper towels to purify DNA and RNA from plants, microbes, blood or mammalian cells in 30 seconds...into this mixture to absorb the nucleic acids. Containinants bind cellulose less tightly than nucleic acids...placing the dipstick into a wash solution removes contaminants while nucleic acids stick around. Finally, purified...

...on the Addgene blog Antibodies 101: Introduction to Immunofluorescence  Antibodies 101: Multiplex Immunofluorescence... al. 2022). So what’s a researcher to do if they want to understand the ultrastructural relationships ...ExM protocols are compatible with conventional antibodies, chemical dyes, and genetically encoded fluorescent...integrity of different targets and labels. You may want to check out a few different protocols to look for...et al (2015) Resolving bundled microtubules using anti-tubulin nanobodies. Nat Commun 6:7933. https://doi.org...microscopy using fluorescent Fab fragment secondary antibodies. Methodsx 9:101796. https://doi.org/10.1016/j.mex...labeled using standard fluorescent proteins and antibodies. Nat Biotechnol 34:987–992. https://doi.org/10.1038...

...reaction (primers, dNTPs, polymerase) using the supernatant of lysed bacteria as template; and 3) run your...colony PCR primers The first and perhaps most important step to colony PCR is designing primers. There...Blunt end cloning is an example of when you might want to know the orientation of the insert. One member... Empty Backbones at Addgene Each approach has advantages and drawbacks, which are summarized in the table...clones for later culture: At this point, you will want to hang onto your clones for later use. There are...to start cultures of your positive clones. If you want to store your clones longer term, just streak the... you choose, make sure to use the appropriate antibiotic for selection.   Lysing bacteria and setting...

... the presence of oxygen during maturation. This meant that the protein could be taken directly from A....however, point mutations are acceptable. GFP's main advantage over conventional fluorescent dyes of the time...to GFP and new iterations of fluorophores are constantly being engineered. Table 1 lists a few common ...wavelength (blue derivatives) Y145F Increases quantum yield for BFP Y145A and H148D Stabilizes the ...uses currently exist, and new GFP technology is constantly being developed! Fusion tagging: One of the ... plasmid. This can serve as an alternative to antibiotic selection. Plasmids of this type may have the...modified ES cells, which can then be used for implantation and generation of transgenic mice. Purification...