...on-site, but now we were presented with a new challenge: automating the bioinformatics steps needed to...input to the next via a Nextflow concept known as channels. Since a Nextflow process can utilize any scripting...process.  Next, we specified the input and output channels for the initial process and tested it by running...number of input file names or filtered the input channel to only use the first n items. To be sure, we went...first, to make sure we understood the concept of channels and implemented the input declaration correctly...faster runtime and decreased cost compared to purchasing and maintaining our own infrastructure. Our patchwork...

...sensitivity Longer protocolIncreased chance of cross-reactivityChallenging to develop assay Competitive...antibodies. Assay development, however, can be challenging since scientists must first identify two independent...Disadvantages Direct Fast and simple protocolReduced chance of cross-reactivity Higher level of background...availability Longer protocolLess specificityIncreased chance of cross-reactivity Sandwich High specificityHigh...Competitive Appropriate for small moleculesReduced chance of cross-reactivity Less sensitivityLess specificityLimited...

... of the fly (Pfeiffer et al., 2010). Yeast (Saccharomyces cerevisiae) Yeast, one of the simplest eukaryotic...as Parkinson’s disease (PD). PD is primarily characterized by protein misfolding that leads to a build-...invaluable in understanding the basic molecular mechanisms underlying the disease (Menezes et al., 2015)... development, cell signaling, aging, and mechanotransduction to name a few. For example, Andrew Leifer....5 (2011): 190-195. Liu, Wei, et al. "From Saccharomyces cerevisiae to human: The important gene co‑expression... PubMed PMID: 26712014. Pandey, Udai Bhan, and Charles D. Nichols. "Human disease models in Drosophila...

...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...

...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...

...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...

...: In one of my favorite books, Michael Ende’s Momo, the title character endears herself to the nearby ...lack thereof): Information sharing gets more challenging the bigger an organization gets, and creating...communication that gets sent out through multiple channels is not reasonable or sustainable. That said, when...village (and to the reader) by simply listening. Chances are, you know someone like this, even if you’re...these people yourself.  Fig 1: Quote from Michael Ende, courtesy of morefamousquotes.com A good...

...may be familiar with popular light-sensitive ion channels used to control activities like neuronal signaling...activity. Researchers have designed a variety of photoswitchable elements into many other types of proteins....under blue light BcLOV4 undergoes a conformational change and binds the plasma membrane under blue light ...The authors demonstrated its use to probe cell mechanics, vesicle transport, and cell division, but given...family signaling pathways. Rho guanine-nucleotide exchange factors (GEFs) activate their partner Rho GTPase...formation, cell contraction, or other morphological changes, depending on the specific Rho GTPase/GEF pair ...

...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...

...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...

... 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...

...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...

...applications from traditional imaging to FRET and stochastic single-molecule super resolution imaging. mNeonGreen...is also insensitive to physiologically relevant changes in pH. Find mTFP1 plasmids here! mWasabi: a bright...mutagenesis approach and found that specific amino acid changes favored blue-shifting more than others. During ...Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging."...blog Resources on Addgene.org Learn about the Michael Davidson fluorescent protein collection Check out...

...master’s thesis in science communication, I had the chance to carry out a comprehensive review of the literature...findings relevant to my background (neuroimaging, biomechanics, neuropsychology) and from areas in which I ... and the tools to deploy its wisdom remains a challenge. Departments like ours reflect a growing effort...display in our building, each day brushing past and chatting with individuals whose research ranged from wildlife...confluence of science and the world it hopes to change. Most researchers are aware of the importance of...

...retrofitting our car with a freezer wired to a charger and two deep cycle batteries for power so the samples... voltmeter on top of the battery box to monitor charge status and a disconnect switch between the battery...spring requiring an overnight hike in. Navigating challenges: collecting and processing environmental samples... Paul and Anastasia (Potanina) Neddersen characterized a few samples by 16S rRNA gene sequencing which...thermostable Cas9 enzymes have previously been characterized: GeoCas9 from the Doudna lab and ThermoCas9 ...

...researchers working on rare diseases, offering them the chance to win some of the latest tools and technologies...They now have some theories on the underlying mechanism, involving the primary cilia. They’ll put a variety...number of stressors, including emotional stress or changes in temperature.  “The disease was first described...a rare disease that can cause hearing loss and changes in coloring of the hair, skin, and eyes. The researchers...syndrome (WS4) by Pediatrician Duangrurdee Wattanasirichaigoon carried different mutations in a gene called...

...was researching, but I decided I needed a new challenge. Looking for sales and marketing jobs Like most... choices we make, my job choices were mostly by chance. Luckily, around the time I decided to look for...have been their target market for many years and purchased their products, so you have some of the best ideas...started with the Harvard Business Review or some Chartered Institute of Marketing reviews, I would have struggled...post-doc a Cambridge University where he studied mechanisms of cell division. Recently Seán got the entrepreneurial...

... to a channel number based on intensity. Higher intensity events are assigned to higher channels. In a...then flow cytometry can help you overcome these challenges and make these types of experiments easier and...of the protein. In some cases this can lead to changes or a complete loss of protein function. In addition...fluorophores Fluorescent antibodies get around the challenges of fluorescent protein fusions by binding to ...stream oscillates to generate droplets that are charged as they pass through a metal deflection plate. ...

...Fluorophore detection Live APC-Cy7 channel eGFP FITC channel CD45 BV421   Figure...of fluorophores from Figure 1. For example, the chart tells us that PE and PE-Cy7 are "very bright" when...conjugated to Cy7, however, (APC-Cy7) the brightness changes to "dim" for the same laser type. Knowing that ...fluorophore, we have to tell the cytometer which channel to use to detect eGFP. As you saw in Figure 4, ...overlaps with FITC, hence reading out the FITC channel in the cytometer's software (without using the ...

...above) include Channelrhodopsin2 (Chr2), a blue-light sensitive non-specific cation channel, activation ...inhibition of action potential generation. Archaerhodopsin (Arch) is activated at a similar spectrum as...reliably locate your injection site. We recently purchased this model ( no longer available through Kopf)...editorial board, Jacqueline N Crawley  [et al]. 2012;Chapter 1:Unit 1 20 1-18. doi: 10.1002/0471142301.ns0120s59...PMC3337962. 8. Zhao S, et al. Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection...

...research projects.   Understanding drug resistance mechanisms of Mycobacterium tuberculosis Dr. Patricia Sheen...her group are interested in understanding the mechanisms behind Mycobacterium tuberculosis resistance ... these proteins will be tested. Investigating changes in neuronal activity after ischemic brain injury... investigates the molecular and cellular etiopathomechanisms of ischemic stroke, the most common type ... Tsakanova to image cortical neurons and study changes in neural activity after ischemic brain injury....

...constructs with AAV. In the case of channelrhodopsins, fewer channel proteins are required for the transduced...is to increase the AAV concentration. In this flowchart, you can see the dwindling proportion of AAV particles...gets transported up the cell’s axon. While the mechanism for this function is not yet fully understood,...S9-S15. PubMed PMID: 11841606. 2. Schultz BR, Chamberlain JS. Recombinant Adeno-associated Virus Transduction...

...necessarily to chat, but to mimic your usual work environment.  Be kind to yourself. This is a challenging period...Receive and provide support while being safe. @GHP_HarvardChan — Shekhar Saxena (@ssaxenageneva) March 16,...give back by supporting local businesses (ex: purchasing a gift card to use later or ordering take-out...exploration site Inventing Heron has a radio reporter challenge, and code.org has coding tutorials for all ages...

...transcriptional changes. Using this road map, and what was known about previously characterized TFs, the Zhang...association of the uncharacterized TFs is a useful finding. Transcriptional program changes from this screen...can be used with other selection and screening mechanisms, although the unique barcodes for each TF help...

...and numeracy on perceived climate change risks. Nature Climate Change, 2(10), 732-735. doi:10.1038/nclimate1547...sources of information if those sources affirm or challenge their beliefs. I asked participants to read short...read an abstract for a study with findings that challenged their beliefs, they were much more likely to ...a phenomenon called “motivated reasoning,” the catchall term for a range of neat little tricks our minds...

... the materials in our catalog are very well-characterized. During this time, we’ve also created a variety...this, throughout the year we rolled out multiple changes to our website, including the recent homepage and... a look at the least visible part behind these changes: the backend technology that powers the new website...granularly customize the search engine, and a scoping mechanism that allows category-specific, narrow searches...among others. We also created a normalization mechanism to “collapse” variations and synonyms of the same...

... virus used. All of these expression control mechanisms require a specific modification of the viral ... years ago. Since then, medical technology has changed a lot, but viruses have stuck around. Vaccine development...Viral vectors are being explored as the delivery mechanism for increasingly popular nucleic acid-based vaccines...specifically AAV, are also being exploited as delivery mechanisms for gene therapy. The tractability of AAV, low...1742-4690-4-66. PMID: 17888184; PMCID: PMC2075523. Bulcha, J.T., et al. Viral vector platforms within the...