...preparations are genome-containing (Figure 1).    Figure 1: Electron micrograph of an Addgene... by the temperature parameters. For example, we found that changing the annealing temperature of the PCR...viral vector solution, and generally these methods measure different characteristics of the solution. These... containing viral genomes. Physical titers are measured by quantifying the concentration of viral genomes... report the physical titer of a viral solution (Figure 2). Because physical titers are used for dosing...values mean and how they can be compared.   Figure 2: Label that comes on Addgene’s AAV aliquots. ...circled in red) report physical titer, which is measured by qPCR.   Physical titer is typically calculated...

... of temperature on total crop production and the production window. A two-way MANOVA could measure the..., you could test the impact of temperature, humidity, sunlight exposure time, and watering volume on total...power. Not sure if your data meets the assumptions of parametric tests? If you aren’t sure if your variance...outliers or data spread within those data sets. Measures of dispersion – standard deviation/error, variance...in a certain direction (i.e., you just want to measure if a sample is greater than a control), then a ...variable. A two-way ANOVA could test the effect of temperature and humidity on an outcome such as total crop... one-way ANOVA can test either the effect of temperature or humidity on crop production.   MANOVA The ...

...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...

... Figure 1: Schematic representations of the indicated unbiased genotyping options. Figure reproduced...other elements, including those relating to DNA structure (e.g. epigenetic modification, bulges) that may...conventional algorithms may not provide a full picture of the impact of CRISPR editing in the model cell...negatives Combining sequencing techniques can ensure validated experiments Unbiased detection methods...sequencing and in silico prediction should give a broad picture of all possible editing events in the genome; from...induced by CRISPR-Cas nucleases in human cells." Nature biotechnology 31.9 (2013): 822-826. PubMed PMID...reveals RNA-programmed Cas9 nuclease specificity." Nature biotechnology 31.9 (2013): 839-843. PubMed PMID...

...downregulation. Figure 3 shows a histogram for a yes-no dot plot, like the one shown in Figure 1.  ...populations in the histogram plot in Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes...Before you read out your plots, you’ll want to make sure you’ve also adjusted voltage and compensated your...adjusting voltage: During data acquisition, make sure that the signal for the positive population is no...should overlap with the negative control population (Figure 1). Based on where the GFP- population ends, you... the total cells present in the gate.   Figure 1: Example of yes-no flow plot of the (negative...expression, low expression, and everything in between (Figure 2). These smears tend to occur when you are looking...

...Nishimasu H, Nureki O, Zhang F. Nature. 2015 Jan 29;517(7536):583-8. doi: 10.1038/nature14136. Epub 2014...Cas9’s crystal structure. “I thought maybe we could use information from the structure to guide and improve...technology. These libraries are lentiviral in nature, so please ensure that you are equipped and authorized to...genes, it is not possible to test each one and make sure it’s working. You can only do that screen with a... the SAM pooled library In a paper published in Nature in December, Konermann and her colleagues in Feng...-scale, knockout screening). As the Zhang lab's Nature paper reports, “Here we have shown that the SAM...Therapeutic uses in humans are also on the table. “In the future, there is potential there,” he says. "We have to...

...the purification procedure. Good indicators of DNA purity are the absorbance measurements at 230, 260, and...same. Before purifying a plasmid from a bacterial culture, it is important to consider your experiment.  ...Cloning grade DNA Transfection grade DNA Figure 1: Transformation introduces transformation grade...range). It can be extracted from small bacterial cultures (i.e. 2-3 ml) with or without commercially available...and DNA for transformation doesn’t need to be as pure as for other uses.   Cloning grade DNA Molecular...also dictate the required sizes of the bacterial cultures and commercial kits you will adopt for the extraction... cloning it is important that the preparation is pure from other nucleic acids (genomic DNA and RNA), ...

... of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes, and... fluorescent labels. In Nature Methods (Vol. 19, Issue 2, pp. 149–158). Nature Research. https://doi.org...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 fluorescent protein...

...unique changes in their spectral properties upon exposure to a specific wavelength of light. PA-FPs can ...fluorescence, to a brighter fluorescent state by exposure to a specific wavelength of light. The switch ...switches from green to red fluorescence. mEosFP exposure to UV light causes an irreversible cleavage near...formation of mEosFP’s chromophore requires a temperature below 30°C and this is not ideal for experiments...emission of green fluorescence, but then further exposure to blue light also inactivates the chromophore...state. Dronpa can be reversibly reactivated by exposure to UV light. The fact that blue light both activates...photons are being emitted, making it easier to capture a clear image above background. The majority of...

... 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...

... 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...

...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...

...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...

...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...

...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...