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...office space on the nicely manicured lawn of University Park at MIT.  I’m sure at some companies, employees...those early years, the founders knew that company culture and enjoying your job were important parts of team... team buidling - they relieved some of the pressure of start up life and maintained their sanity by throwing... still isn't time for goofing off, the company culture here at Addgene has remained paramount. Candidates...Addgenies reply with an enthusiastic, “Our company culture! Addgene is the best place to work, we do SO many...over 60 people join the fun - including a lot of future Addgenies as shown in the photo below.  Kids and... was another successful event! Addgene company culture At Addgene we organically developed the philosophy...

...relief that I attended the amazing, postdoc-driven Future of Research Conference at Boston University last...postdocs are taking some steps to control their own future in science (see my previous blog on this).  Indeed...writing about issues in our science training infrastructure for some years.  He spoke eloquently and vehemently... the need to pay postdocs more.  What will the future of research be? The second day of the conference...conference organizing team.  The topics were Training, Structure of the Workforce, Metrics and Incentives and Funding...beginnings of solutions can’t come too soon.  Watch the Future of Research Website for news and updates on the...

...power of light. In a recent article published in Nature Methods, Boyden and his team describe the discovery... or pathology, and by disabling neurons you can figure out what they are necessary for. We published our...branch on the tree of life. Our most recent paper in Nature Methods offers the first true two-color pair. We'll... to see how a monkey changes decision making, to cure a human patient. We can't do it all by ourselves...Distinct Neural Populations. Nathan Klapoetke et al. Nature Methods. 2014 Feb 9. (PubMed). Read More: Other...

...science in D.C. "Scientists shouldn't be hidden figures.  Ask me about my science!", read my sign.  People...University. She studies beta-cell function and failure in Type 2 Diabetes. Birmingham, AL, USA 33.5207...data collection, destroys existing data, and guts future funding.I marched because science belongs to the... some little scientists (as you can see in the picture) and this march proved to be the perfect opportunity... a Scientific Instrument Maker at Intellectual Ventures Laboratory. Vancouver, BC Canada 49.2827°N 123.1207...attended the march in Vancouver, BC, although I wasn’t sure I’d go until several hours before it started. It...Previow Post on the March for Science Learn How Future of Research is Advocating for Change Within the...

...FPs to highlight various subcellular structures. Please see Figure 1 for commonly used markers for major...up of organs, cells comprise compartments and structures, called organelles. Take a sneak peak inside ...fluorescent protein fusions to visualize subcellular structures. For example, you might use your fluoresent protein...organelle of interest. Click on the gene names in the figure below to find plasmids containing that particular...find more plasmids for labeling your subcellular structure of interest in the Allen Institute for Cell Science...possible to get an idea about the subcellular structures your protein resides in and potentially which...proximity of two proteins within the same subcellular structure or protein complex. By coexpressing both the marker...

...with the literature There are a few ways to make sure you are keeping up with the literature in your field... with the scientific literature a little bit at a time. Searching for literature My go-to search engines...the literature until you have all of the information you need. Reading primary research literature The...papers are also just poorly written. By nature, scientific literature is information dense and since it continually...you daily updates with relevant literature. Organizing the literature As a scientist, you will begin to...require numerous other references to scientific literature to substantiate the facts upon which it builds...means you have to become familiar with a body of literature related to the topic. Finding reliable references...

... manipulate biology based on molecular features found in nature, with the ultimate goal to improve the...impact on this challenge either for magnetic capture or for capture using microfluidic devices. Addgene: What...antibodies against common tumor markers to subsequently capture cancerous cells expressing low levels of tumor ...binding stability really mattered for efficient capture. We wanted to be able to build a highly multivalent...for keeping going while we worked out the cell capture system.) Cancer and other applications Addgene:...technology mean in the context of cancer? Howarth: The capture of circulating tumor cells (CTCs) is going to have...treatment of cancer. It is clear that one needs to capture a diverse range of CTCs to cope with the variation...

...addition of a structured motif to enhance pegRNA stability PEmax — optimized PE architecture; increased ...synthesized (Figure 8A), then cloned in a pooled format into lentiviral transfer vectors (Figure 8B). Libraries...highest levels of single-gene activation (Figure 9D) Figure 9: Overview of CRISPRi/a. In bacteria, activating...Berkeley. Plan Your CRISPR Experiment Resources Figures All figures were created using BioRender.com (Link opens..., N., Ishitani, R., Zhang, F., & Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA...vivo genome editing using Staphylococcus aureus Cas9. Nature , 520 (7546), 186–191. PMID: 25830891 Rauch...changing the target sequence present in the gRNA. Figure 1: Overview of the basic CRISPR mechanism. CRISPR...

...isolated and placed in a buffer. Antibody Structure Figure 1: Structure of an antibody A standard antibody is...Antibodies can also be produced in cell cultures using hybridomas, cell cultures created by fusing B cells with...form a “Y” shape, as shown in Figure 1. The two arms of the Y structure are responsible for binding the...Monoclonal antibodies are generated from cultured B cells, where each culture should produce the same antibody...Diabodies can be used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared... a signaling molecule. Figure 4: A direct sandwich ELISA, which uses a capture antibody and a primary ...In a sandwich ELISA, a capture antibody, bound to a surface, is used to capture the protein of interest...

...Table 1. Figure 4: Third-generation lentiviral plasmids. Created with BioRender.com. Feature Second-generation... chromatin architecture through large-scale mapping of lentiviral integration sites. Nature Protocols ...to continue their lifecycle and replicate. This feature can be harnessed for stable delivery of various... typically ranges from 8–10 kb, encoded on RNA (Figure 1). This RNA is reverse transcribed into the provirus...have been removed or mutated for increased safety. Figure 1: Wild-type lentivirus genome. Created with BioRender.com...largely intact. First-generation plasmids include (Figure 2): Transfer plasmid — contains transgene and wild-type...envelope removed Envelope plasmid — contains env Figure 2: First-generation lentiviral plasmids. Created...

...replication and also act as signals for packaging. Figure 1: Wild-type AAV genome. Created with BioRender.com...do not trigger a strong immune response. These features have made AAVs the preferred choice to be used...components, see the AAV Plasmid Elements table . Figure 2: Plasmids required for rAAV vector production...methods (such as a Gateway destination vector), so be sure to confirm your chosen plasmid is compatible with...available in your lab. When cloning your plasmids, be sure to use recombination deficient bacterial strains... the ITRs found in transfer plasmids. This will ensure that the repeats will be maintained and often results...and instead provided by the chosen cell line to ensure safety and prevent the potential formation of replication-competent...

...elements are summarized in the table below. Figure 2: General structure of a plasmid. Plasmid Element Description...bacterial cultures with antibiotics, see Addgene’s protocol on inoculating bacterial cultures . Antibiotic...for proteins are transcribed and processed into mature messenger RNA (mRNA), which is then translated ...study DNA fragments of interest, such as genes. Figure 1: Creation of recombinant DNA. Working with Plasmids... organisms, including plants, worms, mice, and cultured human cells. Have diverse applications — Although...one unique restriction enzyme recognition site (Figure 2). These elements allow for the propagation of...propagation of these plasmids through cell division. To ensure the retention of plasmid DNA in bacterial populations...

...different optimal editing windows. Be sure to refer to the literature associated with your chosen editor....activity. Be sure to sequence the genomic region you intend to target in your samples to ensure there are...components (Figure 2), and the optimal system will likely depend on your cell type. Figure 2: Different...(Cas enzyme and guide RNA) for your experiment (Figure 1). You will decide how to express Cas9, the delivery... and finally how to validate your genetic edit. Figure 1: Flow chart describing the general framework ...and essentially function as a “knockout,” but be sure to consider potential consequences of having a non-functional...only compatible with smaller Cas enzymes, like S. aureus Cas9. For more information, see our blog post on...

...Collection page . Figure 5: Overview of AdEasy™ adenoviral vector production system. Figure is based on the...assemble icosahedral capsids and build new virions. Figure 1: Wild-type adenovirus and its genome. Genes over...which incorporate additional genomic deletions. Figure 2: First-generation rAdV vectors. Created with ...using first- and third-generation rAdV vectors. Figure 3: Second-generation rAdV vectors. Created with... challenge of the HCAdV vector production is to ensure no carryover from the HV in final viral preparations... genome or using other plasmids instead of HVs. Figure 4: Third-generation rAdV vectors and helper virus...methods (such as a Gateway destination vector), so be sure to confirm your chosen plasmid is compatible with...

...to continue their lifecycle and replicate. This feature can be harnessed for stable delivery of various...gamma-retroviruses ranges from 9–11 kb, encoded on RNA (Figure 1). This RNA is reverse transcribed into the provirus...producing viral vectors in a laboratory setting. Figure 1: Wild-type gamma-retrovirus genome. Created with...gamma-retroviral vectors, you need three plasmids (Figure 2): Transfer plasmid — contains transgene, sgRNA...infectivity for different cell types (known as tropism). Figure 2: Gamma-retroviral plasmids. Created with BioRender.com...methods (such as a Gateway destination vector), so be sure to confirm your chosen plasmid is compatible with...available in your lab. When cloning your plasmids, be sure to use recombination-deficient bacteria strains,...

...and the distal promoter (Figure 4). Figure 4: Basic eukaryotic promoter structure. TF = transcription factor...recognizes different core promoter sequences. Figure 2. Basic structure of a bacterial promoter. TSS = transcription...sense or coding strand of the transcribed gene (Figure 1). The coding strand is the DNA strand that encodes...strand that is transcribed by the RNA polymerase. Figure 1: Simplified promoter region during transcription...positions from the transcription start site (TSS) (Figure 2). The Pribnow box (TATAAT) is located at the ...is an example of a negatively-controlled operon (Figure 3). A positive-repressible operon works in the ...is an example of a positively-controlled operon. Figure 3: Lac operon regulation. Created with BioRender.com...

...guide to channelrhodopsin variants: features, limitations and future developments. Exp Physiol, 96 (1),...social dysfunction. Nature, 477 (7363), 171–178. https://doi.org/10.1038/nature10360 PMID: 21796121 Yizhar...Optogenetics Plasmid Collection . An optogenetics procedure. A channelrhodopsin, fused to mCherry, is expressed... allow the fast depolarization of neurons upon exposure to light through direct stimulation of ion channels... in excitation (depolarization) of the neuron. Feature enhancements include: Increased photocurrent amplitude...from the cryptophyte Guillardia theta ). Other feature enhancements include: Increased photocurrent amplitude...outside of the brain, rendering the experimental procedure less invasive. Different activation wavelengths...

...that form complex secondary structures. Read more in our SLIC blog post . Figure 6: Summary of LIC. Created...Read more in our Restriction Cloning blog post . Figure 1: Restriction enzyme cloning of your gene of interest...conditions. Read more in our Gateway Cloning blog post . Figure 2: Summary of Gateway cloning. Created with BioRender.com...cloning. Read more in our TOPO Cloning blog post . Figure 3: Summary of TOPO cloning. TOPO = topoisomerase...exonuclease. Read more in our Gibson Assembly blog post . Figure 4: Summary of Gibson assembly. Created with BioRender.com...Read more in our Golden Gate Cloning blog post . Figure 5: Summary of Golden Gate cloning. Created with...time- and cost-saver for labs working with yeast. Figure 7: Summary of yeast-mediated plasmid cloning and...

...explore Addgene's Chemogenetics Plasmid Collection . Figure 1: General overview of a chemogenetics experiment...muscarinic receptor and has not been used in vivo. Figure 2: DREADDs, their ligands, and signaling properties...and/or K + ions, and activates neuronal activity. Figure 3: PSAM4-based ligand-gated ion channels, their...optogenetic systems in our Optogenetics Guide . Figure 4: Luminopsins can be activated through chemogenetic... control expression of a chemogenetic reporter ensures that the reporter remains silent until a cell or...Neurosci, 37 , 387–407. https://doi.org/10.1146/annurev-neuro-071013-014048 PMID: 25002280 Strader, C. ...

...can also negatively affect data reproducibility. Figure 1: Simplified flow chart for amplifying and using...infected cell receives only one plasmid. To make sure that every plasmid is adequately represented in ...two types: positive screens and negative screens (Figure 2). Both types of screen employ a selection method...re planning to conduct a pooled library screen. Figure 2: A simplified outline for using lentiviral gRNA...cells that survive post-selection. The selective pressure must be strong enough that most of the cells die...

...conjugated to a signaling molecule, can be used to capture, quantify, and identify proteins of interest. Read...Optogenetics integrates optics and genetic engineering to measure and manipulate cells (frequently neurons) and their...optogenetics research utilize light to detect, measure, and control molecular signals and cells in order...

...Not sure where to start? Need a quick reference? Addgene's eBooks are here to help!... Collection Not sure where to start? Need a quick reference? Addgene's eBooks are here to help! 0 of 0...you need to know about plasmids, from parts, nomenclature, and uses to common promoters and cloning techniques...

... a protein-coding gene, and a terminator, as in Figure 1). Next, up to six Level 1 modules can be assembled...maintain the final plasmid after transformation. Figure 1: Overview of the MoClo Toolkit components and...synthetic biology. MoClo Plant Parts II and Infrastructure Kit Plant Expression Johannes Stuttmann 95 ...

...crystallography cannot capture flexible or disordered regions, so crystal structures like those in Figures 2 and 3 don...biology! Major features First, we’ll cover the basic parts of the Cas9 enzyme.   Figure 1: A cartoon...from Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo state...state that it doesn’t appear in the crystal structure (Figure 2) at all. When this crucial recognition region...learn about this process!      Figure 3: Crystal structure of S. pyogenes Cas9 bound to guide RNA...are disordered and don’t appear in the Figure 3 crystal structure, reflecting the less stringent recognition...tolerate mismatches in this region. The crystal structure in Figure 3 shows the guide RNA stabilizing the DNA-binding...

...contamination of the rest. Avoid long exposure of the cell culture to air and make sure to tighten the flask cap before...contamination in your cell culture or you are about to begin a new cell culture project. If mycoplasmas ...They are present in most of the cell culture facilities, tissue culture labs and every cell culturist has... every cell culture. Reusing the same bottle of serum again and again for each subculture can enhance ...does not ensure complete sterilization as mycoplasma can even escape 0.2-micron filters. Figure 1: Contamination...seen in the culture, it is a good option to discard the flasks (unless the source of culture is irreplaceable... to be 100% sure. Before testing for mycoplasma, the cells should be in continuous culture for at least...

...particular features of your plasmid, it is advisable to check out the genotype of your E. coli to ensure it is... larger culture volume can improve plasmid yield. Our lab often doubles the volume of culture used for...by adding chloramphenicol to the culture medium. Improve the culture media Changing to a richer media ...the given volume.  Let oxygen in! Make sure your culture is getting the right amount of oxygen for optimal...prevent your cultures from reaching the desired density. The volume of your flask or culture tube should...four times greater than your total culture volume and you should ensure the shaker speed is fast enough ...sufficient gas exchange in the overnight culture. Generally, large cultures in flasks may be shaken around 220...

...your incubation temperature. Some proteins aggregate at 95 °C, a common lysis temperature, so consider a...background noise. Temperature and time You may need to optimize your antibody incubation temperature and time....incubations at both temperatures if needed. Washes Check your antibody manufacturer for wash recommendations... for either a shorter (for less exposure) or longer (for more exposure) time. If you’re seeing high background...expose them. You’ll need to make sure the sensitivity of your exposure solution matches the expected amount... variants Check literature Posttranslational modifications Check literature Highly charged...Underexposed Increase exposure time Chemilumenescence: use a more sensitive exposure kit Too much ...

...generally take longer.) Make sure to take a look at the manufacturer’s kit pages as well! Some kits that...variety of manufacturers. It also includes a rough comparison of list price between these manufacturers, as ...you do want to make sure you’re getting the best bang for your buck. If you’re unsure if a kit is a good...CoPurifying plasmid DNA from bacterial cultures is a core protocol in molecular biology. And while kit-free...applications require large amounts of high quality DNA. Figure 1 illustrates the three grades of DNA you may need...on their relative quality and quantity.   Figure 1: Plasmid preps fall into different categories... bacterial endotoxins, that could affect your culture’s viability. Avoid Low Quality/High Quantity – We...

...developing future sensors.   Figure 7: B-gTEMP temperature-sensitive probe enables fast measurement of temperature...with new tools to measure their favorite targets and finding new targets to measure! Let’s take a look...starting point for further engineering. Figure 5: Structure-guided design and genome mining both led ...capable of local temperature sensing with sub-millisecond resolution and sub-degree temperature differences... improved sensors for these and other targets. Figure 1: MTRIA biosensors are generated by inserting ...D, et al. A fluorescent sensor for real-time measurement of extracellular oxytocin dynamics in the brain... Read more from the authors in this blog post. Figure 2: iGluSnFR3 displays enhanced brightness, expression...

...antibodies. Because these mixtures are defined by the manufacturer, each rAb in a mixture can be individually...your target! Incubation temperature and time Incubating at a lower temperature reduces background noise...ll need to lyse and denature them. If you are intentionally running non-denatured proteins, in what is...take multiple exposures for a blot with varying signals on it. It requires long exposure times (up to ...not available in a pure form, you can perform relative quantification, which measures protein amounts and... a lot to cover, so let's dive in!      Figure 1: The technical decisions to make when designing...antibodies, check the manuscript and supplementary figures for validation data, or follow the references to...

...see both of these features in the gating strategy shown in Figure 6.  Figure 6: This flow data,...plot (Figure 1). Clustered events are called a population.                             Figure 1: ...spread apart show a low density of cells (Figure 7).  Figure 7: Contour lines in flow data from Jhunjhunwala...soon find yourself encountering flow data in the literature. Data reported from flow cytometry experiments...single cells in a population. It can be used to measure protein expression, identify rare cells, or even... cytometers, and output is generated through measurement of events. An event is something that passes ..., or A-B+ cells. If you look at the dot plot in Figure 2, you might assume that all cell populations are...

...with two measurements: forward scatter (FSC) and side scatter (SSC). To acquire these measurements, the cytometer... that are being measured: height (H), area (A), and width (W). The different measures are represented ...just as fine (Figure 4). As H and A scale in proportion (while H and W do not; see Figure 2), single cell...sequence of gates to be able to select and precisely measure your cells of interest. In many experiments you... are analyzing a homogenous cell population in culture, it is normal to observe diversity in cell size...cell at a low angle (FSC) or large angle (SSC) (Figure 1). FSC values depend on the cell's size, while... allow you to adjust this setting as needed. To figure out the appropriate voltages to observe your cells...

...of neurons is for more than making pretty pictures (see figure 4). It also enables single-cell anatomical...particles and labeled a variety of colors (figure 4b). But notice in figure 4b how difficult it is to trace individual...transduction, measured via GFP expression, was determined 3 weeks later. Table was derived from figure 2e in ...transduction, measured via GFP expression, was determined 3 weeks later. Table was derived from figure 3b in ...diversity is maintained at both high (figure 4e) and low doses (figure 4f) of the tTA inducer virus, but ...expression in both the CNS (figure 5 in Chan et al) and the PNS (supplemental figure 8 of Chan et al). These... Two new AAV capsids (figure 1) were engineered with the CREATE method (figure 2) to allow for efficient...

...based on Fuguo Jiang’s structure of Cas9 – primed for cleavage. This structure captures the displaced non-...space, but their flexible and jiggly nature suggested the dynamic nature of a protein. The Bender’s popularity... the 3D structure of these proteins, it is necessary to download a pdb file of the structure’s atomic ...inflexible plaster. As a result, the model obscures the flexible nature of all proteins in general, and the ...one. Why do we need physical models of protein structure? Structural biologists have always struggled to...effectively communicate the nuances of protein structure in a flat 2-D images. In the early days of structural...to bend 1/8th inch steel wire into a backbone structure of a protein, based on its atomic coordinates....

...commonly used to measure relative quantification of mRNA, which can be used as a proxy measure for differences...on hairpin structure included in primer Yes Yes Agarose gel Table 1: Features of various...primer variable is the annealing temperature, not the melting temperature. Though you’ll want to predict...the presence of primarily denatured (single-stranded) DNA. As the temperature drops, single intensity should...Thellwell, 2000). See Table 1 for a comparison of features of several commonly used chemistries)   Chemistry...gels Scorpions Low Probe is on a hairpin structure included in the 5’ primer Yes Yes. Agarose...ll want to stay under 150 bp long Annealing temperature should be 60-65°C For qPCR, the important primer...

... interrogated by the optical system (Figure 1).   Figure 1: Cells expressing a particular surface...into progenitor B cells, immature B cells, plasma cells or others.   Figure 2: Due to differences ...preparation, staining procedures, and controls. To prevent instrument clogs, ensure that samples are single...Have you ever wanted to measure expression of your protein of interest in a single cell? Or perhaps, ...specific subset of cells in a population in a procedure termed fluorescence activated cell sorting or ...combines three systems to analyze single cells from a mixture (3): An optics system A fluidics system An electronics...flow cytometers detect Cell size and complexity, measured using visible light scatter The optical system...

...standard GenBank feature types. Clicking on a particular feature here also highlights that feature in the “Map...to find the plasmid with the features you need quickly. SnapGene’s Feature Library - SnapGene’s software...these features, such as Cas9, weren’t detected by our in-house software. In fact, SnapGene’s feature library...Annotated Features, Enzymes, and Primers - The new maps have the identities of most features labeled directly...and “Sequence” tabs. Features The table found here lists the common features detected in the plasmid...SnapGene’s sequence viewer software and extensive feature library, our updated plasmid and sequence displays...seamlessly identifies a variety of common plasmid features such as antibiotic resistance genes, selectable...

...at 260 nm (A260). Figure 1: Characteristic absorption spectra for pure DNA and likely contaminants...A260/230 values that are close to those of pure DNA (Figure 1D). This property means that even samples...Do you know how much plasmid DNA you have or how pure your sample really is?  To assess the concentration...Microvolume spectrophotometers make it quick and easy to measure sample concentration and purity using only a tiny... the sample itself — so easy that this simple procedure is often taken for granted. In this post, we take...vary.  How it works — briefly Spectrophotometers measure how light travels through a sample — in this case... wavelengths with maximal absorption at 260 nm (Figure 1A). Thanks to the Beer–Lambert law, the instrument...

...make sure that the sample is pure and free of any contaminants that could affect the measurements. You'll...based on its measured response. An example of this is shown in Figure 2.   Figure 2: The concentration...allergy labs use standard curves with ELISAs to measure allergen concentrations in food; and your friend...dilute a control sample of known concentration and measure a specific response as a function of the concentration...the BCA reagent. The absorbance of both is then measured on a spectrophotometer and concentration versus...series diluted by the same factor. For example, Figure 1 depicts a 2-fold dilution series. Each step of...optimize your standard curve accordingly.    Figure 1: A 1 mg/mL control is diluted in a series ranging...

...make sure you have sufficient volume to dip all your plants. Make sure your Agrobacterium cultures are ...transformed agrobacterium culture Dipping your plant’s flowers in the agrobacterium culture to allow for tDNA ...transformant. Remove mature siliques prior to transformation: Siliques that are mature prior to transformation...volume of culture you chose to work with. You will get Agrobacterium all over your gloves, so be sure to change...transformant takes a long time. Make sure the agrobacterial culture you are growing definitely contains...If your cultures have been incubated at stationary phase for too long, the cells in your culture will no...antibiotic-containing media) A similar process occurs in nature; Agrobacterium tDNA insertion likely precipitated...

...India at room temperature. Partnering with our distributor allowed us to ensure that temperature-sensitive...kept at the appropriate temperature for the duration of their journey. Figure 2: Annual distribution ...from that country. Stats and figures are helpful, but our favorite measure of our educational resources...from our repository to an amazing 111 countries (Figure 1)! But while we were making the materials available... the accessibility varied country to country.   Figure 1: Number of countries Addgene distributes to per...”: a distributor who, among other things, could ensure our packages navigated customs and arrived at their...destination in a timely manner and at the correct temperature. To date, we have partnered with six distributors...

...the age of your culture post thaw. In general, the higher the passage number of a culture the lower the ...growth, or clumping. To ensure the best possible results for any tissue culture-based experiment, labs ... & Chen, T.R. “Mycoplasma infection of cultured cells.” Nature 339 (1989). 487–488. PubMed PMID: 2725683...packaging cells and your target cells. Poor tissue culture practices can lead to low titers and poor transductions...transductions. Cell lines should be routinely subcultured at densities that are low enough to prevent confluency... high enough to promote cell growth. Overgrown cultures stimulate quiescence, the resting phase of the...the transfection and transduction efficiencies. Cultures should only be passaged 20-30 times before discarding...

...secondary structures in your pegRNA could inhibit editing (Anzalone et al., 2019). Figure 2: Starting...disrupting its canonical structure and Cas9 binding (Nishimasu et al., 2014). Figure 3: pegRNAs whose ...genome, ensure it is not homologous to the target genomic sequence (Chen et al., 2021). Figure 8: ..., N., Ishitani, R., Zhang, F., & Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA...RT) template including the desired edit. Figure 1: Prime Editing schematic. Created with BioRender.com...this location bind to Cas9 with canonical sgRNA structure (right). Created with BioRender.com. What ...lead to indels (Anzalone et al., 2019). Figure 4: Editing the PAM prevents the prime editor from...

...cryo-EM grid.     Figure 1: MagIC-cryo-EM for sample enrichment and structure determination. A) Schematic... this post: MagIC-Cryo-EM for enrichment and structure determination LEA proteins protect samples during...astrocytes MagIC-Cryo-EM for sample enrichment and structure determination By Mike Lacy Conventional cryogenic...that combines enrichment and imaging of proteins captured on magnetic beads (Arimura et al., 2024). In MagIC-cryo-EM...GFP fusion in this proof-of-concept study) are captured on streptavidin-coated magnetic beads through ...and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment streamlines the purification...bundle; SAH: 60-nm single alpha helix) and target capture module (SpyTag-GFP nanobody) assembled on beads...

...Then, mix the PCR products and use temperature to denature and renature the dsDNA. Upon renaturation, you...confirm your results. (Figure 5). The digestion product (Figure 4, lanes 0.3 % 3.0, Figure 5, lane 2) is faint...expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal cell...comparing it to the undigested control (Figure 4, lane 0, Figure 5, lane 1) is what confirms it’s a real...Cas9 lentiviral vector, lentiCas9-Blast (52962-LV, Figure 1). Some cells were then used to generate a monoclonal... The rest of the cells were frozen down. Figure 1: Cas9 expression in A549 cells transduced with... cell pool had wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects...

...into the appropriate stab culture Grow the stab culture Ship the stab culture to the proper location To...Every single step in a lab procedure can and will fail. Some step failures require re-attempting the same...do when something fails. While we’re sure our techs could figure it out if they needed to, they’d have...interested to see which part of her procedures are the most failure prone. In an academic lab, a similar...data for lab procedures. With this data in hand, we can go back and optimize our procedure to make the ...To keep ourselves from mixing up samples and to ensure that everything is grown successfully, we must ...following code examples show how software can be structured to allow for the messiness of biology. These ...

...particles. Direct measurement of lentiviral RNA is one alternative to direct p24 measurement. Like a p24 ELISA...that specific feature. Primers targeting the transgene are also beneficial as they ensure that the correct...stable cell line. While we’ve all experienced the pressure to move a project forward, transductions should...time to properly titer your virus will not only ensure that your infection is designed in the best possible...flavors, physical or functional. Physical titers measure the amount of viral particles in a sample and are...acid. Functional titers, or infectious titers, measure how many of the viral particles produced can actually...common assays for physical titer are direct p24 measurements via ELISA and qPCR for viral RNA. Measuring ...

...blot.   Total protein measurements as loading controls Total protein measurements are an accurate alternative...control measurement will take place either before or after blotting for your POI. Figure 6: Total...the most important feature of a loading control, and both total protein measurements are linear. Note the... a western blot requires a standard curve with a pure sample of your protein of interest (POI). This approach...pipetting or irregular membrane transfer. Figure 1: A loading control is a type of positive control... and the resulting fraction is reported. Figure 2: Normalization with loading controls. Under each...experimental design and gel quantification. Figure 3: In a perfect world, protein concentration in...

... to the versatility of the basic antibody structure (Figure 1). Antibodies can be broken up into smaller...purpose. Figure 1: Overview of antibody engineering methods. (A) Basic structure of an antibody.... sections. Structure-guided directed evolution If you desire a bit more control, structure guided directed...is the modification of antibody sequences and structures to affect their function. Usually, the goal is... PCR, degenerate primers, gene shuffling, and structure-guided directed evolution. The nice thing about...they all follow the same general cyclical process (Figure 2). The differences typically come in when generating... the starting point for the next cycle. Figure 2: General process of antibody directed evolution...

...other materials have been washed away (Figure 1).      Figure 1: The steps of rAb production. Created... make sure to look up the setting for your isotype.  Antibody concentration may also be measured through...antibodies with some molecular biology and cell culture experience. Let’s go over the basics of making ... days (in mammalian cells) to collect the cell culture supernatant for further processing. It may be helpful...appropriate immobilized ligands are incubated with cell culture supernatant containing antibodies. The antibodies...your antibody in a downstream step, you’ll want to ensure that your buffer components are compatible with...have an IgG protein setting. If yours does not, be sure to set the absorbance to 280 nm (Pace et al., 1995...