...Stochastic multicolor labeling is a popular technique in neuroscience and developmental biology. This... persistently fluorescent during development. Stochastic multicolor labeling systems, many based on Brainbow...even axons of up to 100 microns in length. Some challenges of the Brainbow system are that it does not use...for improving the level of detail possible for stochastic multicolor labeling of the brain. Labeling Drosophila... researchers who developed Tetbow. Conclusion Stochastic multicolor cell labeling systems allow single...Harbor Protocols 2011.1 (2011): pdb-prot5546. Richardson, Alexander, et al. "Visualizing the Fate of Transplanted...

...genome editing without a nuclease Although the mechanism of incorporation is not well understood, researchers...double strand break repair, it has proven more challenging to develop high efficiency CRISPR-based approaches...Schofield, 2015; Moser and Hirsch, 2016; Epstein and Schaffer, 2017) Hopefully you’ve been able to check out...research currently focuses on determining the mechanisms and consequences of cohesin gene inactivation...27280971. 10. Epstein, Benjamin E., and David V. Schaffer. "Combining engineered nucleases with adeno-associated...Medicine, CRISPR, and Genome Engineering. Springer, Cham, 2017. 29-42. PubMed PMID: 29130152. PubMed Central...

...to simultaneously test how multiple amino acid changes affect a protein of interest’s function. This technique...combination and possibly discover unexpected epistatic changes in protein activity. See Natarajan et al for an...mutagenic primers to introduce all desired codon changes (step 1 in the image above). A second universal...This results in the final product having a codon change present on both strands of the plasmid. The ssDNA...in your research? References 1. Natarajan, Chandrasekhar, et al. "Epistasis among adaptive mutations ... Mccullum, E. O., Williams, B. A., Zhang, J., & Chaput, J. C. (2010). “Random Mutagenesis by Error-Prone...

...nature. Once the bond is formed it is unaffected by changes in pH, temperature or organic solvents. Uses of...it is added to. Biotin also has an exposed side chain that can be easily manipulated to make reactive ...vivo. Biotinylation reagents Biotin’s exposed side chain can be cross-linked to amino acid functional groups... terminus of a DNA sequence during a Polymerase Chain Reaction (PCR) by using a biotinylated primer. Biotin...substrate (Holden and Cremer, 2003). References 1. Chapman-Smith, A., & Cronan, J. E. “Molecular Biology of...129 (2009a): 477–484. PubMed PMID: 10064313. 2. Chapman-Smith, A., & Cronan, J. E. “The enzymatic biotinylation...

...and increased tariffs present an unprecedented challenge to the scientific research ecosystem, not just...concerned about these developments. These abrupt changes threaten to slow transformative biological discoveries...years to come. We Are Here to Help Despite these challenges, Addgene remains steadfast in our mission to ...resilience and creativity in the face of funding challenges. By strengthening our commitment to collaboration... invite researchers to share how these funding changes are impacting your work and what support would ...

...known about bioluminescent organisms and their characteristic radiance for some time – there are records ...after that, in 1994, Prasher’s collaborator Martin Chalfie expressed GFP in exogenous organisms (E. coli, ...and later C. elegans) for the first time. After Chalfie’s work, scientists really started to take notice... Roger Tsien resulted in much of our current mechanistic understanding of GFP’s function. His lab described...Nobel Prize in chemistry was awarded to Shimomura, Chalfie, and Tsien for their work on GFP. This great honor...

...alterations in gene expression, but causality and mechanisms remain unclear. Today, targeted epigenetic modification...could identify the target genes of previously uncharacterized enhancers. The popular TALEN-based LITE system...H3K27ac histone modification at specified loci. Charles Gersbach’s lab has deposited mammalian expression...sorting and selection of transduced cells. Grant Challen’s lab also created constitutive (pCMV-dCas9-D3A)...publication at Addgene. Maeder ML, Angstman JF, Richardson ME, Linder SJ, Cascio VM, Tsai SQ, Ho QH, Sander...Fowler T, Rees R, Kramer A, Martens A, Edwards JR, Challen GA (2016) Reprogrammable CRISPR/Cas9-based system...

...Magenta. Or you can use Color>Split Channels and then Color>Merge Channels into whatever combination of colors...antibody Secondary antibody SCN1A sodium channels anti-pan-Nav R-mAb K58/35R (Mouse IgG2a) Goat... (Mouse IgG1) Goat anti-mouse IgG1 Kv2.1 channel anti-Kv2.1 KC (Rabbit IgG polyclonal) Goat ...images of each labeled target as different color channels and analyze the results. Depending on the goal...rather than red-green). Even better, show each channel in individual panels, then the multicolor overlay...

... technology was invented to introduce specific changes into the mouse genome. With these early technologies...Mice. My first CRISPR mouse experiment Everything changed with the advent of CRISPR in 2013 (Cong et al.,...CRISPR-Cas9 is an acquired immune system in bacteria and archaea. As you know well if you’ve been following the ...sends out an “SOS” signal and cellular repair mechanisms rush in to repair the damage. If all they can...form of an oligonucleotide or plasmid), precise changes can be made in the genome via the homology directed...repair pathway (HDR), be it a single nucleotide change, insertion of a reporter gene, or replacement of...

... some challenges during my internship, but nothing could have prepared me that those challenges would ...always been interested in the ways that I could change the world around me. My strong interest in the ...exploring my different interests but also a way of challenging what I am used to. Brandon Solis, marketing...structured career because our mindsets and needs can change. Having been at Addgene for a month, I can say ...

...Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins by Cary Valley, Shreya Vedantam, Michelle...fluorescent proteins at Addgene. 7. Designing Your Chalk Talk for the Academic Job Interview by Erik Snapp...blogger Erik Snapp breaks down how to prepare a chalk talk for an academic job interview including defining... research vision, organizing your vision into a chalk talk, and tips for practicing.  6. The Fluorescent...insertions, deletions, and all possible base-to-base changes.   1. Plasmids 101: Transformation, Transduction...

...from Zygosaccharomyces rouxii), B2 (from Zygosaccharomyces bailii) and B3 (from Zygosaccharomyces bisporus...as low toxicity. FLP/FRT recombinases (from Saccharomyces cerevisiae) had been used successfully in Drosophila... Kotelevtseva N, King K, Davis JR, Mullins LJ, Chapman KE, Dunbar DR, Mullins JJ. Nucleic Acids Res. 2007...

...selection. Knock-in events tend to be rare, and the challenge with any experiment is in efficient recovery of...increase in knock-in efficiency, presumably by channeling DNA breaks into the homologous recombination ... template design The Meyer lab meticulously characterized several aspects of editing. First, they devised...injected animal.  PAM sequence considerations For changes 5’ to the PAM, sequences corresponding to the same...Prize included Jennifer Doudna and Emmanuelle Charpentier for their pioneering CRISPR/Cas9 work (now recognized...proteins associated with a given genomic locus (CRISPR-ChAP-MS) could further transform the range of experiments...

... suggest changes to our social media strategy. We ended up implementing some of these changes and seeing...my current projects and personal interests. We chatted about social media analytics and summer travel ...at once. I was able to do something that was challenging while helping us share our resources better with...video production, and keeping inventory of our merchandise collection. While most days I was up to my neck...

...Matthew J. Niederhuber, a graduate student at UNC Chapel Hill. Chromatin immunoprecipitation followed by...interactions, the reliance on fixation does increase the chance of false-positives, as proteins may appear to be...rely on nuclei extraction. They report that this change offers a significant improvement and simplification...especially in situations when it is technically challenging to collect large numbers of cells or when a high... student at the University of North Carolina in Chapel Hill, where he studies enhancers and blogs about...

...blogs to keep them current. Here are some of the changes we’ve made: New CRISPR plasmid pages We’ve given...but with far fewer off-target effects! This characteristic probably explains why pC016 - LwCas13a guide...RNA targeting systems, RNA editing does not make changes to the genome, thus providing an avenue for temporary... Expanding CRISPR Beyond SpCas9 Day 8: Repair mechanisms: CRISPR 101: Non-homologous End Joining and PITCHing...podcast with Addgene Advisory Board member Alex Chavez) Day 11: Pooled libraries: Genome-Wide Screening...

...take over. I like writing and science, so I took a chance.” It’s been six years since Hans took that gamble...science talk). I do illustrations for summarizing mechanisms and applications that are relevant to things ...favorite thing to work on. “It’s one of the most challenging [parts of my job], so making it a success feels...colors draw people in. Drawing a picture of a mechanism that also looks nice is a great way to bring people...posts. And make sure to subscribe to the Career Channel of the blog so you don’t miss the next two posts...

...conformational change in the GPCR, leading to the activation of the Gα subunit. The activated Gα exchanges bound...constructs, localization of the Rluc8 was more challenging. The Roth lab used structure-guided protein engineering...activation by drugs to further understand their mechanism. These plasmids are available as a collection,...Slocum ST, Che T, Gavin AC, McCorvy JD, Roth BL, Strachan RT (2020) TRUPATH, an open-source biosensor platform...

...Mask: How Leaders Spark and Sustain Change, by Peter Fuda and Richard Badham Leadership Insights for Engineers...Stages of Your Career: Doer, Manager, Leader” by Charlotte Beers, divides workers into three cycles: Doers... are many good resources on the skills and characteristics of successful leaders. Below I've listed a ...Leadership (Articles on Leadership, also can be purchased individually) ...

...systems, modifications to the RNA components, and challenging target genomes, such as the malaria-causing Plasmodium...how much more there is left to do with the characterization and implementation of this system. Monday ...Addgene depositor, discussed some of the many challenges of creating biological circuits, and I can only... are taking the "genetic code" to a new level, changing it from a puzzle to decrypt to a tool to be harnessed... tools – both deposited and distributed – have changed so dramatically in the past 4 years. I kept busy...