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...neurotropic variant of AAV9, is better than AAV9 at gaining access to the CNS of C57BL/6J mice following IV...6J mouse strains likely contributed to the unintentionally selection of a virus with mouse-strain dependent... to select novel AAV capsid variants could unintentionally limit the utility of candidate capsids.  Are...Ken Y., et al. "Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous...

... feeling of camaraderie one gets from a well-functioning lab team. Obviously not everyone gets this feeling...environments outside of academia provide a formal training regime and the right set of tools to help you ...non-scientists! Maybe you’ve just spent 5-10 years learning to “speak science,” and virtually every person... years of focusing on my technical skills with cloning and lentivirus production, I didn’t really start...

...are typically lysed in a non-denaturing buffer containing non-ionic detergents, such as NP-40 or triton-X...nonspecific proteins from the sample prior to beginning the IP.  During the IP, the target protein in ...Immunoblotting.) For mass spectrometry, elute in a urea-containing lysis buffer. Alternatively, the protein can ...control, confirming that the capture antibody is functioning as expected. In order to identify any non-specific...

...truth is, there is a lot of software development happening at Addgene under the hood. A significant portion...moment a deposit kit is sent to a depositor, to streamlining the order fulfillment process to reduce viral...language and it took us a total of two years, from planning to completion, to do this upgrade while continuing...principles for open science. I hope you enjoyed learning about Addgene’s software development team and ...

...antibacterial drugs. Resistance can be intrinsic, meaning a basic characteristic that naturally occurs in...especially if the infection is serious or life-threatening, treatment will often begin before this information...results. While this is not the only method of determining drug resistance status, it is a very common, ...essential component of preventing infection, as is cleaning and disinfecting frequently touched surfaces like...

...immortalized human cell line. He proved this by examining whether the cell lines had isoenzymes and genetic... piece of paper on the front of the incubator containing the passage number, cell line, and date, which...He studied biochemistry at Warwick University, gaining extensive cell culture research experience through...Cell Culture Guide Thermofisher Guidelines for Maintaining Cultured Cells Invitrogen Cell Culture Basics...

...of control plasmids Part of planning your experiment includes determining what factors need to be controlled...independent variable you are testing and not some other unintended factor. Control plasmids are used to minimize...systems have many variables. However daunting designing and executing these steps may be, proper controls...

...of E. coli for DNA propagation. While great for cloning purposes, these E. coli strains are not usually...Use BL21 (DE3)   Basic IPTG-inducible strain containing T7 RNAP (DE3) F- ompT lon hsdSB (rB- mB-) gal... (DE3)** Rifampicin RecA-deficient; allows for cloning and expression in same strain F- recA1 hsdR(rK12...most experiments, the lac promoter can “leak,” meaning that a low level of expression exists even without...

...backbone. This step requires “counter-selection,” meaning that the selection is for the loss of a specific...protein. This allows conjugation between a pir-containing donor and the recipient of interest. Because ...transcriptional readthrough into the MCS to facilitate the cloning of toxic products. To allow manipulation of naturally...But if they are blue or magenta, they are real! Planning an allelic exchange experiment? Find the pTOX ...

...protein. These were either (i) coded by the multiple cloning site and therefore consist of random amino acid... often use restriction enzymes in the multiple cloning site for generating fusions. For the POI-fluorescent...PVAT, when we use the AgeI site in the multiple cloning site) is sufficient. For the fluorescent protein-POI...immunofluorescence or properties of the protein.  Determining other aspects of functionality (catalytic activity...

...might be considered a double-edged sword with unintended consequences. In this blog, we’ll take a deep...the name suggests, these lentiviruses lack a functioning integrase, the enzyme responsible for inserting...homologous recombination or non-homologous end joining (see Figure 2).  Figure 2: Schematic representation...interruptions. Insertional mutagenesis is especially concerning in clinical settings, as evidenced by retroviral...

... Chalk Talk These items should be part of the beginning of your chalk talk. These should be plainly stated... implicated in several human diseases. Proper patterning of cells and tissues is fundamental to development...you understand what it takes to be successful in running a lab, getting grants, and writing papers? This... and earn some cash for my expensive habits of running shoes and single malt whiskies, but I remember ...

... primarily by two pathways: Non-Homologous End-Joining (NHEJ) and Homology Directed Repair (HDR). NHEJ...your site of interest, and a donor DNA molecule containing your desired edit are all you need to generate...generate a knock-in.   General considerations for designing a repair template to create mutations HDR donors...Featured Topic Page Learn about Non-Homologous End Joining Learn about Base Editing Read 3 Tips to Improve...

...essential for proper muscle function. Without functioning dystrophin, an individual experiences progressive...skipping proceeds through the non-homologous end joining (NHEJ) pathway. It’s estimated that very little...Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. In vivo genome editing using...Used to Edit Genes Read How Non-Homologous End Joining is Used to Delete Genes Additional Resources on...

...Stauffer, W. R., Fish, K. N., & Pfenning, A. R. (2022). Machine learning sequence prioritization for cell...cells in C57BL/6J mice, but not in BALB/cJ mice, meaning that systemic capsids that rely on LY6A interactions...2017). AAV-PHP.eB has frequently been used for screening candidate regulatory elements such as enhancers...capsid of choice for future promoter/enhancer screening. However, AAV-PHP.AX packages viral genomes at...Gradinaru, V. (2017). Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous...

... the cell, mitochondria play a vital role in maintaining cellular function. Mitochondria can also lend...capacity of cancer cells. This is certainly just the beginning for MitoTRACER, as it is a diverse tool that can...converting white adipose tissue (WAT) cells to brown or browning adipose tissue (BAT) — a type of fat tissue that...researchers developed a version of the kit that uses melanin production, allowing students to explore cell metabolism...

...cell (Dudek et al., 2018; Korneyenkov & Zamyatnin, 2021), meaning what works great in one system or cell...could help, or harm, your experimental goals when designing and testing your AAV.  Figure 2: Comparison...10.1016/j.omtn.2017.06.011 Korneyenkov, M. A., & Zamyatnin, A. A. (2021). Next Step in Gene Delivery: Modern...

... could try. We hope this post helped you start planning for your next multiplex IF experiment! If you ...Resources References Andrews, N. P., Boeckman, J. X., Manning, C. F., Nguyen, J. T., Bechtold, H., Dumitras, ...doi.org/10.17504/protocols.io.bs68nhhw Im, K., Mareninov, S., Diaz, M. F. P., & Yong, W. H. (2019). An ... Introduction to Performing Immunofluorescence Staining. In W. H. Yong (Ed.), Biobanking (Vol. 1897, pp...

...they are the result of multiple plasmid copies combining in a process known as plasmid multimerization.... multimers grow more slowly compared to those containing the monomeric version of the plasmid (Summers...Multimers Run an undigested plasmid on agarose gel Running undigested DNA on a gel as a control when performing... 1984; Summers et al., 1993). In these cases, screening multiple bacterial colonies using one of the detection...

... the exciting new experiments that you’ve been planning, it is a good idea to pause and make sure that...approach may not be best if you are ultimately planning to use your antibody to detect endogenous levels...be a factor in your analysis. Example: You are planning an RNA-immunoprecipitation experiment and want...tuned for ready-to-use antibodies from Addgene Designing a recombinant validation experiment? Find plasmids...