...type of interest. Repeat rounds of infection and screening by PCR enrich for capsid variants with increased...single-cell anatomical study and neuronal tracing, aka determining where a neuron’s axons and dendrites extend to...brain, you needed to make a transgenic mouse. Combining AAv.PHP.eB’s or AAV.PHP.S’s great neuronal transduction...Yoo BB, et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous...

...your studies.  Find the plasmids to set up your screening systems at Addgene! Schematic of the evolution...- there's a simple method for that! By Michelle Cronin The benefits of nanobodies over conventional antibodies... secreted into the supernatant. Four plasmids containing different nanobodies (NB) were generated in pMAK... dual-deaminase base editor enables concurrent adenine and cytosine editing. Find the plasmids at Addgene...

...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...

... 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...

...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...

...E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F (2016) C2c2 is a single-component programmable...Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F (2017... L, Joung J, Konermann S, Severinov K, Zhang F, Koonin EV (2015) Discovery and Functional Characterization...Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 Is a Single RNA-Guided ...

...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 ...

...managing a lot of references. I cite as I write, meaning I use the software to add the citations in real...write. Things are going to get a little crazy (meaning you are probably going to cite hundreds of references...better to keep your references organized from the beginning. I also recommend using the citation style of ...relevant to you, note it, and move on. At the beginning I felt like this was cheating. Eventually I learned...

...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...

... and Olsson, 1971), plant lectins (wheat germ agglutinin WGA, Schwab et al., 1978) or fluorescent molecules... mapping of the neuronal circuitry involved in learning and memory (Kohara et al., 2014; plasmids at Addgene...Gradinaru, V. (2017). Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous... motor to auditory pathway important for vocal learning. Nat Neurosci 20(7), 978–986. PubMed PMID: 28504672...Javoy-Agid F., and Agid Y. (1978). Labeled wheat germ agglutinin (WGA) as a new, highly sensitive retrograde tracer...

...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 ...

...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...

... 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...