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...applications.   Figure 1: Live-cell labeling strategies. The green structure represents a fluorophore... end up linked to whatever molecule you choose (Figure 1). Just express the fusion protein and add a specific...fluorophore or other useful ligand, gray structure is the protein of interest, and black is the fusion tag ...-tag construct can help you better detect dim structures or low levels of expression with the same imaging...super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and... enables applications like multi-color imaging (Figure 2). If you’re working with purified proteins in...colors depending on the needs of your experiment (Figure 3). And using self-labeling tags makes updating...

... alert the day of the procedure. Mice move much more quickly than cell cultures do, and tired researchers... some nerves. Following safety procedures and practicing safety culture, however, can let you work with...risks and how to minimize them. Built-in safety features   When viruses were first being engineered as ...still enter (and thus infect) cells, this safety feature prevents the virus from replicating and spreading... including yourself as the researcher.   Figure 1: Separation of retroviral packaging components...precautions to take, and if something does happen, ensure you can receive appropriate treatment and/or monitoring...monitoring. Virus-specific concerns   In nature, some viruses are more dangerous than others, and the...

... to ensure all plasmids pass our high-quality standards and contain the expected plasmid features.  ...based on SnapGene’s “Common Feature” library — with some additional features added by the QC team — and...the plasmid to ensure our assembly result is accurate The gene/insert or any major feature highlighted by...Nobody’s perfect! This is a platitude I’m sure we have all heard. It’s a truth that extends to both people...optimizing experiments, to false conclusions.  Figure 1. Potential points where DNA errors can be introduced... Figure 2. Addgene’s basic QC process. Created with BioRender.com. Once Addgene receives deposited...resolving a variety of QC issues. This process ensures that any potential sequence discrepancies can be...

...also stable at this temperature and can bind to the primers.  Extension- The temperature is then raised slightly...slightly to Taq polymerase’s ideal temperature (70-75oC). At this temperature Taq polymerase can synthesize...and the use of a fluorometer feature built into the thermocycler to measure that fluorescent output. With...resistant enzyme that can withstand changes in temperature. Taq was first identified in the late 1960s during...separation of the two DNA strands.  Annealing- The temperature is then dropped to a range in which the forward...forward and reverse primers are stable. At this temperature the primers can anneal to the single stranded...the target DNA quickly and accurately.  Figure 1: Steps in PCR reaction   This process of ...

...present in the sample (APC in the case of Figure 1). In Figure 1A, the population needs to be shifted towards.... Let's look at an experimental example in Figure 2. Figure 2A shows the sample prior to compensation....the populations move, as in Figure 2, following compensation. Figure 3: Flow plot for a double positive... a compensation value between the two channels (Figure 1A). Applying compensation means you are telling...values later in your analysis, unlike voltages.  Figure 1A shows a single-color APC control, with APC and... a straight line and are only positive for APC (Figure 1B). For single-color samples, you can only change...would decrease the compensation value.   Figure 1: Compensation example with single color control...

...your own figures Google Drawings Google Drawings is similar to using PowerPoint to draw figures. It’s part...useful for drawing figures with lots of elements. It took some playing around to figure out all the settings...project my team & I have poured our hearts into for 2 yrs) has 200K+ figures by 95K+ scientists 👨🏽‍🔬...when writing posts. Since then I’ve realized my figures are just as important, if not more important, than...drawing tools, so it’s harder to draw intricate figures, like a brain or a mouse. Vectr Vectr is like a... Vectr to be fairly intuitive and I was making figures quickly. Inkscape Inkscape is the most similar...Illustrator out of the three options for drawing your own figure discussed in this post. It’s open source and available...

...design goals. Examples:  “feature aware”, “SNP aware”, “secondary structure aware”, and “microhomology...collective utility, it’s easy to fault the lack of features in a single application. The question is then,... solve, and finally how we might proceed in the future. When we think of designing experiments using CRISPR...have broken down the tools into the individual features that make each tool unique. The CRISPR Software...Software Matchmaker is composed of these features and enables you to select the tool(s) based on your project...advanced functionality, it seems these kinds of features are delegated to other more suitable software ... removing repetitive tasks and/or by providing features to help with the post-design process such as primer...

...activates halorhodopsins to silence neuronal activity (Figure 1) (Wiegert et al., 2017). How to get an opsin...into the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable...promoter only in specific neuronal cells. FLEx ensures, therefore, spatial accuracy and strong opsin expression... functional topography of the neural circuit architecture of the brain regions associated with motivated...on developmental biology at the École Normale Supérieure de Lyon. Her love for educational science led...genetically targeted optical control of neural activity." Nature neuroscience 8.9 (2005): 1263. PubMed PMID: 16116447...: PMC2820367. Deisseroth, Karl. "Optogenetics." Nature methods 8.1 (2011): 26. PubMed PMID: 21191368. ...

...codon optimality reveals hidden signatures of cotranslational folding." Nature structural & molecular biology20.2...but some possible reasons include: Metabolic pressures - it takes cellular resources to produce tRNAs...Protein folding - If a protein is encoded by a mixture of codons with highly and poorly charged tRNAs,...codons are associated with specific secondary structures in 10 closely related yeast strains. Adaptation...properly. You might then think, “Okay, I’ll just make sure the abundances of the codons I choose for the host...Angov et al., 2008), but there are still many more features to consider when designing a full gene. A non-...Restriction sites Sequences prone to create secondary structures in RNA transcripts Effects on transcription (...

... and in vitro applications.   Figure 2: Modular features of DIRECTED particles where the fusion...-the-art. PE6b was very efficient for short, unstructured templates and significantly reduced undesired...efficient at complex edits, such as long or highly structured templates. Cas9 variants PE6e–g often had even...varied depending on the specific edit.   Figure 1: Choosing the best PE6 variant for your application...type-specific delivery by modular envelope design. Nature Communications, 14(1), 5141. https://doi.org/10.1038...therefore been trafficked between locations). Figure 3: A) LOV-Turbo becomes active when illuminated... control of proximity labeling in living cells. Nature Methods, 20(6), 908–917. doi: 10.1038/s41592-023...

...short-read sequencing are highlighted in Figure 2. Figure 2: Overview of short-read sequencing technology...pnas.74.12.5463  Shendure, J., & Ji, H. (2008). Next-generation DNA sequencing. Nature Biotechnology, 26... that’s not the only technology Addgene uses to ensure accuracy of deposited plasmids. To resolve QC issues...recognized and recorded as a DNA strand is synthesized (Figure 1). Sanger sequencing requires four separate reactions... “trace” file to determine the sequence. Figure 1: Overview of Sanger sequencing technology. Created...by synthesis, but modifies the original Sanger procedure to improve throughput by increasing the number...puzzle pieces together. Once they have the full picture of the puzzle, the QC team compares it to the plasmid...

...repository, but be sure to let us know if you'd like to write about your plasmids in a future blog post. No...found at Addgene and in Figure S12 of the publication. Agudelo D, et al. Nature Methods. 2017. PubMed ...Rees H, et al. Nature Communications. 2017. PubMed PMID: 28585549 Kim Y, et al. Nature Biotechnology. ...GABA-ergic interneurons here. Dimidschstein J, et al. Nature Neuroscience. 2016. PubMed PMID: 27798629 Listen...Find PhoCl plasmids at Addgene. Zhang W, et al. Nature Methods. 2017. PubMed PMID: 28288123 Listen to...used as tools to learn about the influence of structure on an FP’s properties. pBAD-LSSmCherry1 is a long... Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169  pJL-SaBE3  SaCas9 (NNGRRT)    85170...

...CRY2PHR-activator fusion floats freely, depicted in the figure below. Once triggered by blue light, the CRY2 and...31X. By using a slit pattern during blue light exposure (470nm), the researchers showed that expression...created a photoactive Cas9 tool, as shown in the figure below. Their most successful design utilized Magnet...on and off. As one might expect from the modular nature of Cas9, Nihongaki et al. showed that it was possible...reach deeper tissues. Additionally, high intensity exposure can cause DNA damage. Far-red light, however, ...to create photoswitchable spCas9, dCas9, and S. aureus Cas9 (saCas9) proteins, showing the versatility... variant of AcrIIA4 (CASANOVA). As shown in the figure below, in the absence of light, Cas9 is bound by...

... of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes, and...visualization of both organic tissues and cell cultures, opening biological interiors to many inquisitive... and fluorescent ligands. Figure created with BioRender.com. Fluorescence refers to the emission ...Fluorescent dyes and ligands vary in their chemical structure, leading to differences in their excitation and...ability to retain fluorescent intensity during exposure to light, is another factor to consider. If your...your application requires longer periods of exposure, you’ll want a more photostable fluorophore. Factors...recruitment to receptors, indicating their agonistic nature. The potency and efficacy of these ligands can ...

...guest bloggers Shravanti Surest from Iowa State University. Shravanti Suresh is a PhD candidate who investigates...This post was contributed by Shravanti Suresh from Iowa State University. Since its appearance, SARS-...19 and some serological tests to determine past exposure. However, the limited availability of reagents... turn to other technologies like CRISPR. Figure 1: An overview of the general schematic of CRISPR...with streptavidin, the lateral flow strip first captures the streptavidin portion of the probe via biotin...probe that is cut will continue to flow and be captured by an antibody further along on the strip. Highlights...Isothermal amplification is done at one constant temperature, eliminating the need for thermocyclers. This...

...primary is conjugated to a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence...confocal   Figure 2: High-level overview of an indirect immunofluorescence procedure with key considerations...within a cell,” “Does this disease change the architecture of my cells,” or “How does this mutation impact...non-fluorescent visualization methods. Stay tuned for a future blog post to learn more about these techniques!.... For this strategy to work, it is important to ensure that you are using secondaries with fluorophores...divided into the several steps outlined below and in Figure 2. For a deeper dive into the steps and techniques...involve multiple smaller steps that all help to ensure that antibodies can access their target and that...

...a separate fix/perm cytosolic procedure in that case.     Figure 3: For intracellular labeling...antibody through the biotin-streptavidin interaction (Figure 1a). This interaction brings the fluorophore in...plasmids and biotin plasmids at Addgene!     Figure 1: Indirect detection of target markers can be ...(“anti-rabbit”) region of the primary antibody (Figure 1b). As above, the advantage of this system is ...which would label everything but the CD8+ T cells (Figure 2). Even better, we can use the same fluorophore... gating on the negative population.     Figure 2: Dump gating labels all unwanted cell lineages...membrane but inside the cell. Due to their chemical nature, antibodies cannot penetrate the cell membrane;...

...Symposium and Workshop, organized by Future of Research, Academics for the Future of Science, and the MIT Graduate... More information can be found at http://futureofresearch.org/advocating-for-science-boston-2016/. Sign...Science Symposium Boston 2016 Changes since the Future of Research Symposia Ongoing challenges facing...et al, PNAS 2014; Taylor, eLife, 2016). Several Future of Research (FoR) symposia held across the USA ...science to date. Advocacy opportunities for the future No matter your opinion on these changes, it is ...institutional responses to the FLSA ruling. Similarly, from purely a salary perspective, while this is a move in the...For those advocating in this area, keeping the pressure on for further increases will be challenging, ...

... notes on how lab procedures were actually performed, potentially curtailing future confusion (and hours...community-editable fluorescent protein database ReFigure: Save Scientific Figures into Dashboards and Share Your Insights...reproducibility issues in the biological sciences and featured speakers from academia, industry, nonprofits, ...reproducible if its findings can be predictably applied to future work. Importantly, no one should expect that every...Failed attempts to apply irreproducible results to future work cost both time and money with estimates reaching...doing an experiment wrong. However, even simple procedures like pouring a plate or running a gel have intricacies...initiatives to create more reliable reagents for future research. I’ve talked to many researchers who have...

...factors to consider when choosing one.     Figure 1: Example of an experimental optogenetic setup...activity or gene expression. Experimental output is measured and quantified. Created with BioRender.com. ...either excite or inhibit neuronal activity upon exposure to light within a specific range of wavelengths... be used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light...engineered receptor that change shape with light exposure to activate them. PORTLs use a similar concept...when a signaling pathway is unknown.      Figure 3: Chimeric OptoXR incorporating rhodopsin undergoes...that may go awry in a disease state.      Figure 4: Example of an intracellular signaling pathway...