...incubation at 37 °C. Wash the membrane for 5–10 minutes 2–3x in fresh wash buffer such as PBS or TBST. Stringent...Purpose Tris HCl 62.5 mM Buffer SDS 2% (w/v) Detergent Β-mercaptoethanol (βME) 100...minutes at 50 °C. Wash the membrane for 5–10 minutes 2–6x in fresh wash buffer such as PBS or TBST. Note ... tool to identify a protein of interest in a complicated solution like cell lysate. But they can be a ...

...AAVs in the clinic Thus far, AAV vectors AAV1, AAV-2, AAV1-AAV2 hybrids, AAV-6, AAV-7, AAV-8, AAV-9 and...neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the... 10.24 (2003): 1999-2004. PubMed PMID: 14566358.  2. Hirsch, M. L., et al. "Self-complementary AAV mediates...passionate about HIV cure research and science communication.     References 1. Gaspar, H. B., S. Howe, and...affinity-purified receptor-targeted viral vectors." Nature communications 6 (2015): ncomms7246. PubMed PMID: 25665714...our AAV Production Protocol Check out our AAV Purification Protocol ...

... of components, such as your CRISPR enzyme or Pol 2 promoter. The options available in each class, or ...overhang specific to the type of module it is (Pol 2 protomer, Cas protein, ect...). Whatever individual... (KO, CRISPRa, CRISPRi, editing)   Figure 2: Selecting module fragments on the Fragmid website...ampicillin resistance cassette and an origin of replication, which are not schematized. Sizes of fragments...five months. When they tested those clones (in duplicate), 93% had the correct banding pattern in the restriction...correct. Because they created the assemblies in duplicate, they were able to confirm that at least one plasmid...

...PMID: 27272384 Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et al. "Genome-wide...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674...mismatches on modification by AsCpf1 (red) or LbCpf1 (blue). Mismatch positions are indicated below each ...eclipse any of the CRISPR nucleases in AAV-based applications. Get More Cpf1 Background Here THE ORIGINAL ... off-target editing rates, determined using sophisticated genome-wide analysis. At many of its computationally...compared the ratio of total off-target to on-target modification for AsCpf1 and LbCpf1, and found that both orthologs...

... characteristics of select AAV serotypes in Table 2 from the paper. Delivery of AAV into the brain can...routes for neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest: Cell-type specific...experiments! Late last year, we hosted our first AAV Education and Development workshop aimed at brainstorming...even further. For example, injecting AAVs at one location can result in expression of an optogenetic payload...shown in Figure 1, injected retrograde AAVs at one location result in opsin expression in their cell bodies...

...virus 2 (Mayo-Muñoz et al., 2018). Only viral particles that have the gene deletion could replicate when...base pair to another base pair at a specific site. 2. Temporal, spatial, or conditional control of CRISPR...Editing of Sulfolobus islandicus Rod-Shaped Virus 2. Viruses 10:695 . https://doi.org/10.3390/v10120695..., 2018). For example, AcrIIA4 mentioned above indicates that it’s the fourth Acr protein discovered that...B, Bondy-Denomy J (2020) Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies....light-inducible Cas9 activity? Check out these these plasmids Catch Up on Your CRISPR Background with Our Guide Pages...

...portion of our careers. In that time we may have only 2-3 formal supervisors that will provide guidance and...be challenging – is advice and guidance from only 2 people enough?  For many years I’ve been organizing... helps you reach your goals Listens well and communicates clearly and openly Gives honest feedback without...lab. If only every science trainee had such a dedicated person guiding the start of their career. “I want...

...the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed AAV-68544, we were...look for potential contaminants. The analysis is a 2-step process using Geneious software: First, the individual...These impurities cannot be removed during the purification step because they are inside the virus itself...very low concentration. So how do we make sure to catch all of these potential mishaps before distributing...Guerin et al., 2020).  In addition to genome identification, we developed custom open-access Python scripts...

...3 plates at different densities for selection). A 2% agarose solution is then melted (and thereby sterilized...96-well assay plate. We generally freeze at least 2 ampules each of 4 clones with different levels of ...of agarose, which is then overlaid with the firmer 2% agarose gel solution discussed above for picking....to rapidly punch out cell clones with sterile truncated pipet tips. Indeed, an experienced operator can...wiped down with 70% ethanol, and a box of sterile truncated pipet tips (USA Scientific #1011-8410) is also..., and making sure we always maintain a backup replicates of each clone in case of contamination. We do...

...resolved loci with physical map distances of 75 and 2 Mbp, with the calculated fluorescent distances correlating...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the... its nuclease activity, catalytically dead Cas9 (dCas9) has many applications, the most well-known being...characterizing the 4D nucleome. Future modifications and applications Ma et al. developed this technique ...have major applications to cancer, for example, the visualization of chromosomal translocations or chromosomal...plasmids from this publication at Addgene. A protein-tagging system for signal amplification in gene expression...technique published every week! One of the newest applications is a colorful system that uses fluorescently...

...literature? Very often the data of interest are in 1 or 2 figures from one paper and then another figure in ...ReFigure by creating ReFigures and answering this 2 minute survey! You can also find ReFigure on Twitter...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...

...groups, such as their vaccine production, SARS-CoV-2 research, or adenovirus packaging cell lines dataset...-Line Knowledge Resource. J Biomol Tech. 2018; 29(2): 25-38....compounds, links to publication references, and cross-references with cell line catalogs and collections....surveying of cell line collections websites and catalogues Literature searches utilizing Google Scholar ...include accession number, species, and assigned category (cancerous, hybridoma, etc.), as well as sex and...manually going through the pdfs of the 26,000 publications cited. If the information seems incomplete or...

... tag than protein), 2-3 tags is increasingly common. Tandem affinity purification (TAP) once referred ...TEV cleavage site (more on that in a moment), and 2 ProtA IgG-binding domains. TAP has since come to encompass...found in this paper (available at Addgene).   Table 2: Protease recognition sites commonly used with tags...or DYKDDDK 1 Affinity and Purification Good for antibody-based purification; has inherent enterokinase...site GST Large Protein 26 Purification and Stability Good for purification with glutathione; protects...used for purification as elution requires low pH poly His HHHHHH  0.8 Affinity and Purification Very small...GKPIPNPLLGLDST   1.4 Affinity and Purification Good for antibody-based purification Combo and cleavage tags...

...with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore...nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara Y,... of Visualized Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs details... size and frequency [3]. There are several applications in which the production of deletions by paired...useful to study haploinsufficiency. Subsequent modifications at the remaining allele within the deletion ...

...like this: Figure 2: Expression of Plasmid B (as internal control) This result indicates that the transfection...20074371. PubMed Central PMCID: PMC2817684. 2. Replicates and repeats—what is the difference and is it...down expression of Gene X but, as shown in Figure 2, this was likely due to the original transfection ...two types of replicate controls: technical and biological. In general, technical replicates can be thought... employ transfection, negative, positive, and replicate controls.  Transfection controls: empty vector...the interrogation of the independent variable. Replicate controls: technical and biological Reliable results...derived from one source. Conversely, biological replicates ARE independent and can be thought of as "reproducibility...

... qPCR is that the results can vary by a factor of 2. This means that if you set up two identical assays...amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have a clear separation between...as low as 1C/s. Increasing the elongation time to 2 minutes and the denaturation time to 1 minute has ...take to achieve a clean NTC: Have a dedicated bench with a dedicated set of pipettes for ddPCR set-up. .... Each droplet contains the ingredients for amplification of the target DNA. This partitioning reduces...detection of the product. As an added benefit, replicates are built into the technology. One well (containing...droplet contains the material required for a mini amplification reaction to take place.  When the PCR is complete...

...resistance to the herbicide chlorsulfuron (Homo line 2) or wild type (WT), two weeks after herbicide was ... CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression... when associated with microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule FRET...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration... an enhanced pU6cm promoter, viral replicon amplification of cargo, and heat treatment.  Figure...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...and more responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5 can be imaged...

...properly exiting the ER and fluorescing (see Figure 2) [5].  When using FPs containing two cysteine residues...compartments including the secretory pathway (Figure 2), the inner membrane space of mitochondria and chloroplasts...PMID: 18228466. Pubmed Central PMCID: PMC2875081. 2. Snapp, Erik Lee. "Fluorescent proteins: a cell biologist's...compartments undergo significant post-translational modifications including glycosylation, disulfide bond formation...are equally susceptible to these non-native modifications, which can all potentially affect function [...N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering the protein... non-optimized parental proteins. Our recent publication illustrated the quantitative increase in fluorescent...

...HEK-293T Sf9 Time to collection 2–4 days 5–10 days Purification Ultracentrifugation (AAV); centrifugation...Centrifugation Titration time ~1 day (AAV); ~1 week (lenti) 2–10 days Initial titers Higher Lower Resources... a factory to produce adeno-associated virus type 2 vectors. Human Gene Therapy, 13(16), 1935–43. https...so most labs opt to purchase adenoviruses from dedicated adenoviral production labs or companies. Production...something you may have to optimize for your specific application. With mammalian systems, it is standard to do...more cells, while AAV can be titrated following purification using qPCR or digital droplet PCR. Insect cells...to collect your viral supernatant. Baculovirus replicates inside insect cells, and its lifecycle ends in...

...upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry) to...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong... Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents...Deniz, et al. "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...and Jinfang Zhu. "Immunologic applications of conditional gene modification technology in the mouse." Current...contributed by Alessia Armezzani, scientific communication manager at genOway. A few decades ago, the brain...École Normale Supérieure de Lyon. Her love for educational science led her to pursue a teaching career, ...