...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...

...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...

...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...

...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...

...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...

... 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...

... simple hierarchy: class to type to subtype to variant. Despite the seemingly complicated steps to arrive...on main function and composition; subtypes and variants are typically based on species of origin. There...currently two classes, six types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification...hierarchy flows from Class to type to subtype to variant (shown in italics). Created with BioRender.com.... to as Cascade (CRISPR-associated complex for antiviral defense) complexes, while Class 2 use single protein...after binding to a target. Class 1 is the most abundant of the two, comprising about 90% of identified...others have become more notable in recent years. A variant of V-U, paradoxically called V-K, is a CRISPR-associated...

...are easily studied in living cells using antibodies. Antibodies could further be used to interfere with...screens have been used in several settings, and are fantastic tools for unbiased discovery. With these screens...number of cells needed is lower, and perhaps most important, targeting all genes doesn’t necessarily add value...cytokine known to cause the up-regulation of an important surface protein (e.g. PD-L1 on a cancer cell)....are migratory, and their exact localization is important for their function. Their migration is induced...with the identified surface proteins, and such antibodies could thus be interesting drugs. Known drug targets...in Green Listed.  Provide the list of genes you want to target with your screen in the “Input” box. ...

... are more likely to consider cover letters an important part of the application. Even within an organization...core skill, considers cover letters to be very important. On the other hand, our app/dev team rarely, if...that organization or group. This is particularly important for labs and nonprofits, who tend to prioritize...under thirty seconds.” That’s exactly what you would want to talk about! You could describe your organizational... research], and have excellent [list the most important skills for the position]. I am enthusiastic about...about [something that you love and is important for the job]. Notice that the intros above allow you ...include a brief summary of why you are a good applicant. If you haven't talked about it yet, it's also...

...that you want to use for your experiments and verify that your construct is functional. Advantages of the...more about the Gateway cloning method and its advantages. An introduction to Gateway technology The Gateway... clone and destination vector carry different antibiotic resistance markers (indicated here by plasmid...coding sequence. If you choose this strategy, it’s important to include the proper protein expression elements...Clone When making the expression clone, it is important to choose the destination vector that best fits...Gateway cloning kit, and MultiSite Gateway plasmids. Constant reading frame When you move a DNA fragment from...stays in frame. High efficiency The positive (antibiotic) and negative (CcdB) selection markers used for...

...inconclusive and unreliable. Proper controls provide the constant variables that enable the correct interpretation...of the independent variable you are testing. Importantly, they demonstrate the functionality of your experimental... the transfection reagents and process. It is important to note that optimal experimental conditions, ...Control (mentioned above) could also serve as an important negative control. In this context, the empty vector...interpretation of the results because it provides an important reference point when analyzing the specificity... same time. Although the replicate control is important, it speaks to the consistency of the reagents ...controls are more time consuming and ultimately more important. Ideally, each biological replicate should use...

...highest quantum yield donor, the highest absorbing acceptor and fluorophores with significant overlap ... theory, is an easy technique, but it is very important to follow some simple rules to avoid common pitfalls...purpose of your studies. These parameters are less important when designing a biosensor, as the distance between...and their spatial orientation will be fixed. The quantum yield of the donor (number of emitted photons per...extinction coefficient of the acceptor (linking the quantity of absorbed light, at a given wavelength, to the...Clover Green Mammalian Expresses Clover (a GFP variant) commonly used with mRuby2 pLSSmOrange-N1 Orange... mRuby2 Red Mammalian Expresses mRuby2 (a RFP variant) commonly used with Clover pGWF1 Cyan & Yellow...

... in March, he and his colleagues took another important step forward by dissecting that S. pyogenes protein...that SpyLigase can be used to link affibodies or antibodies against common tumor markers to subsequently ...cancerous cells expressing low levels of tumor antigen. I asked Howarth to tell us more about SpyLigase... to another peptide tag. Addgene: What is the advantage of peptide tags for building larger protein assemblies... shouldn’t get in the way much. Particularly we wanted to be able to build assemblies through covalent...stability really mattered for efficient capture. We wanted to be able to build a highly multivalent assembly...approaches to improve CTC capture, but I hope that our antibody tentacles may have an impact on this challenge...

...then collects the perfusion liquid for quantitation. The advantage of this method is that it can be performed...dopaminergic neurons in a brain region called the substantia nigra, which is the main source of the body’s...inserted into the region of the brain where DA quantitation is desired (Figure 1). The probe perfuses liquid...performed in a live awake animal by surgical implantation of the probe into a specific brain region. While... sensor. This sensor was engineered from a GFP variant and a DA receptor such that GFP fluoresces when...when the receptor is bound by DA. Two variants of the sensor, dLight1.1 and dLight1.2, exhibit high (submicromolar...GRABDA sensor based on a similar strategy: a GFP variant bound to a DA receptor, such that the GFP fluoresces...

...meeting participants. They were the ones holding poster tubes (or as our baffled flight attendant called...internal motto for scientists who contact us and want a guarantee that their experiments will work (yes, this...through TALENs and CRISPR-Cas nucleases, provided important context for the rest of the day. He was followed...curious about these newest features of our website.  I want to offer a huge thanks to all the Addgenies who ... helped get me to this meeting. And of course, I want to thank the organizers of this meeting who have...share their cutting edge science in increasingly important field.  Interested in Addgene's Keystone Poster...

...for synaptic glutamate response. They generated variants optimized for different applications (v82 has ... and powerful high-throughput screen to select variants based on performance metrics specifically for ...vivo (Farahani et al. 2022). The authors take advantage of the SH2 domain of ZAP70 (ZtSH2) which binds...sensors Whatever your analyte of interest, it’s important to choose a biosensor that has a binding affinity...BeadScan platform for screening candidate biosensor variants for performance under two-photon fluorescence ...lifetime imaging. B) LiLac responses are highly resistant to pH changes. Image adapted from Koveal et al...tdTomato), they produce a temperature sensor that significantly outperforms previous designs. B-gTEMP is excitable...

...from their evolution. Southern blotting and quantitative real-time PCR Southern blotting, named for its...DNA identity, size, and abundance. If a scientist wants to know if an organism has a particular gene mutation...commonly used. In all cases, the result can be quantified by the strength of the signal in comparison to...experimental questions. Real-time PCR detects and quantifies a gene or DNA sequence of interest by recording...compare different DNA sequences to see which is more abundant in a sample. Whereas Southern blotting is labor...of high-quality DNA, real-time PCR has several advantages including easier automation, higher throughput... similar experimental questions (see below). Importantly, although their role has changed, restriction...

...optimized, it’s important to ascertain how long the rescue phenotype will last, and more importantly, if it does...genetic disease in a postnatal mouse model, an important proof of concept for future preclinical and clinical... time, which would prevent the accumulation of unwanted mutations over time. DMD is a good choice for ...does not cause obvious germline modification, an important finding given the controversy associated with ...great step forward for CRISPR gene therapy, it’s important to realize that DMD is a simpler case than other...and large animal models with mutations in human-relevant exons 45-55, rather than in the traditional exon...Potential off-target effects in muscle, as well as unwanted germline editing, must be rigorously examined ...

...G418-selected clones. However, there are important disadvantages to the AAV-based approach that must be ...this post. Adeno-associated viruses (AAVs) make fantastic gene delivery vehicles for episomal gene expression...downstream of a functional promoter (gene traps). The resultant gene targeting system could be used to routinely...non-sequence specific frameshift mutations through aberrant double strand break repair, it has proven more...endogenous epitope tags, or the introduction of dominant mutations of a gene into the endogenous allele...nucleases In an effort to combine the respective advantages of the AAV and CRISPR-based approaches, several...generating human somatic cell gene knockouts using recombinant adeno‐associated viruses." Nucleic acids research...

...cells more resistant to chemotherapy. You can find these plasmids at Addgene. Another advantage of mice ...researchers: the mouse (Mus musculus). Mice have many advantages as a mammalian model organism for scientists ...nervous system. The fly has also emerged as an important human disease model with potential for therapeutic...in the fly (Pandey and Nichols, 2011). A huge advantage of using the fruit fly is the array of genetic...due to their simplicity, yeast have become an important experimental model in the study of neurological... 1960s, the zebrafish has gradually become an important model organism. They share about 70% of their ...Craig Mello a Nobel Prize in 2006. One of  major advantages of C. elegans is that they are transparent throughout...