We narrowed to 888 results for: URE

...Download ReFigure extension and visit ReFigure.org. You can reach the ReFigure team at refigure@refigure.org...You can use ReFigure to coherently save analyses with the relevant figures from a literature review. No...currently supported by ReFigure beta) you can include it in a ReFigure. ReFigures are good for you and ...support ReFigure by creating ReFigures and answering this 2 minute survey! You can also find ReFigure on Twitter...reading scientific literature? Very often the data of interest are in 1 or 2 figures from one paper and...performance as in this antibody ReFigure Analyzing replications as in this ReFigure about whether Natronobacterium...The ReFigure extension also tells readers if a paper they’re reading has been linked in a ReFigure making...

...experiment. Figure 2: Comparing CAPTURE and CAPTURE 2.0.     Original CAPTURE CAPTURE 2.0 Biotinylation...between original CAPTURE and CAPTURE 2.0 methods.   Applications of CAPTURE 2.0 CAPTURE 2.0’s increased.... Find plasmids for CAPTURE 2.0! When compared to the original CAPTURE, CAPTURE 2.0 had a ~14-fold increase...Recently the Xu lab developed CAPTURE 2.0, an updated version of CAPTURE that detects chromatin interactions...Regulatory Elements: The original CAPTURE method The original CAPTURE method requires the creation of a.... From here, the CAPTURE method is similar to other 3C methods: crosslinking captures chromatin interactions...next-generation sequencing. Figure 1: Overview of the key steps of the CAPTURE method. After protein and...

...Cell Culture Guide Thermofisher Guidelines for Maintaining Cultured Cells Invitrogen Cell Culture Basics...Outreach Scientist Nick Amiss. Cell culture in the present day Cell culture is a widespread tool used in the...surrounding cell culture over the last few decades some pharmacologists have tended to avoid cell culture in favour...artefacts. The life science literature is a litany of false interpretation in cell culture for this reason. Unreproducible... conduct high quality cell culture. Biorepositories around the world ensure that the cell lines deposited...Type Culture Collection aka ATCC where I work. It is virtually impossible to work in cell culture, microbiology...benefited most as cancer cells are amenable to culture in vitro. Virologists too have benefited from the...

... causes this culture shock and how to overcome it. What causes the lab → office culture shock? Working...her (3 weeks in and there's a big office vs. lab culture shock). I'm looking to see if there are any resources...help with the transition.” The lab-to-desk-job culture shock is a real thing. I experienced it, and many...in an academic research lab involves a few key features that can be difficult to replicate in other work...provided a few counterpoints to some of the causes of culture shock above, but after polling some Addgene colleagues... possible. It’s in their best interest to do so, sure, but it also eliminates that feeling of floundering... need to be in the lab that long because of the nature of your time course, but you’ll inevitably have...

...15 minutes before you start any tissue culture work and make sure that clean air is flowing in. The vent...contaminated. Make sure cells don't stay out too long While you’re preparing things, make sure your cells don...Colloids and Interfaces. Here are some tips for cell culture that will hopefully help you keep a well organized...in-flowing air is normally visible and you should always ensure nothing is covering it. Turning on the UV light...microorganisms that it strikes directly. UV light exposure is also harmful for eyes and skin so keep the ...while work is in progress.Learn more about cell culture contamination in this post from the ATCC Declutter...the laminar air flow around your work area. Make sure to only bring required materials into the hood. ...

...double-stranded DNA cleavage. Nature 533:420–424 . https://doi.org/10.1038/nature17946 Lenay C (2000) Hugo De... on the depiction of genome engineering in pop culture SciFi is one of the most imaginative and most important...fiction. It has always served as a vision of the future and to make us reframe our perspective of the current...in the field of health sciences, energy, and agriculture, its portrayal in popular fiction has been largely...editing without double-strand breaks or donor DNA. Nature 576:149–157 . https://doi.org/10.1038/s41586-019...MacLeod CM, McCarty M (1944) STUDIES ON THE CHEMICAL NATURE OF THE SUBSTANCE INDUCING TRANSFORMATION OF PNEUMOCOCCAL...A•T to G•C in genomic DNA without DNA cleavage. Nature 559:E8–E8 . https://doi.org/10.1038/s41586-018-...

...specific guidance on grammatical errors, sentence structures, and overall format (fonts, font size, legibility...the perfect opportunity to paint a comprehensive picture of your academic interests, potential, and drive... True! But you create a network of friends, and future colleagues that can help pull one another up while...

...registration please go to http://futureofresearch.org/boston/. The Future of Research Symposium A year ago...daily lives and future prospects. Among the topics discussed, many related to the structure of the biomedical... overall structure of the biomedical research sector, and what a tenable workforce structure may look ...biomedical graduates take in their future careers. To participate in the Future or Research Symposium Boston...The second Boston Symposium on the Future of Research will be held from 22-24 October. This blog has ...has been contributed by guest blogger and Future of Research Symposium organizer, David T. Riglar PhD. Here... of the six panel discussions to be held at the Future of Research Symposium Boston 2015 – Academic Data...

...upcoming "Future of Research" conference. You can register for the conference here. The Future of Research...colleagues in envisioning the Future of Science. Attend the Symposium on the Future of Science: A postdoc-...science to be happy. What do we have to change to ensure that all scientists can have successful careers...postdoc-organized symposium on the future of the scientific endeavor October 2-3, 2014 Boston University Jacob...

... Elite: Nobel Laureates in the United States. Free Press. New York. Resources MDH CUREs Community (MCC...number of honorees to have trained under a previous laureate (Zuckerman, 1977; Ma et al., 2020). Many have ...trainees at an earlier point in their careers? A CURE for Biochemistry Undergraduate biochemistry courses... Course-based Undergraduate Research Experience (CURE). Having incorporated a discovery-based lab on planarian... a stable, easily purified protein with easily measured activity, MDH is particularly amenable to in-class...    Fig. 1: Undergraduates in Provost’s CURE prepare to analyze their recently purified MDH to...the impact of phosphorylation on the enzyme’s structure, function, and interaction with other proteins...

...protein variants. The future of DNA feature identification What’s next for common features? We plan to continue...creating a database of common features, and devising rules for identifying a feature even when the match is ...is imperfect. SnapGene's feature database The source of common features was our collection of popular ...identity when detecting a reference feature. For a coding sequence feature that may be used to make fusion... into the original literature, some of it decades old, to provide reliable feature annotations. Even with...that a feature is missing from our database, or if you see an opportunity to harness common feature detection... planning, visualizing, and documenting their procedures. Preventable errors in the design of cloning ...

...Addgene recently attended and sponsored the Future of Research (FOR) Boston Symposium 2015. I personally...takeaways I got from the symposium. The goal of the Future of Research Symposium On the FOR website, the FOR...prevent us from identifying problems with the structure of the academic research workforce, and we are...only 14.3% of PhDs in the life sciences end up in tenure track position 5 years after receiving their PhDs... needs to be asked going forward is: How do we measure inclusion and equity? This is a difficult question...capturing, quantifying, and preventing this type of failure. Final thoughts The FOR Symposium Boston 2015 shined...Stephan’s Book - How Economics Shapes Science The Future of Research Website Additional Publications of ...

...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...

...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...

...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...

...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...

...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...

... view is excited at one time and captured in a single camera exposure. This is much faster than relying...the Harvard Center for Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University...an x-ray, or a biologist imaging the smallest structures of life; the key to a great image is contrast...relatively thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner. This is accomplished...resolution) limit of a light microscope. Due to the wave nature of light, an infinitely small point of light will...improvement in the resolution of a light microscope (see Figure 1). What microscopy technique should I use for ...live cells with fluorescently labelled, motile structures These samples are thin enough to sit within the...

...proteins with a membrane-impermeable biotin substrate (Figure 1). Biotinylated proteins were isolated, identified...shared proteome of astrocytes and neurons (CS SPAN) featured extracellular matrix proteins, cell adhesion molecules...transporters, ion channels, GPCRs, and more.  Figure 1: Workflow for cell-surface protein labeling and...Huntington's disease model) will be valuable resources for future studies of the molecular bases of astrocyte-neuron...recipient cells from red to green fluorescence (Figure 2). Donor cells express iCre with a mitochondrial...DsRed-STOP to trigger expression of EGFP. Figure 2: Schematic of the MitoTRACER system. NLS = nuclear...The power and potential of mitochondria transfer. Nature, 623(7986), 283–291. https://doi.org/10.1038/s41586...

...perform the IP at 4oC. When choosing the capture antibody for IP, be sure to choose one that recognizes the ...target protein/capture antibody intact. The wash step is repeated several times to ensure non-specific ...complexes, wash away unbound protein, elute and measure the target. However, the two different approaches...process of anchoring an antibody, often called the capture antibody, to agarose or magnetic beads in a way...immunoglobulin, leaving the antigen binding sites free to capture the target protein. The beads can be collected ...Agarose beads are sponge-like and vary in shape and structure. Their porous surface provides a large area for...protein interactions, and limit high throughput procedures. However, agarose beads are often a more cost-effective...

... your culture should always be derived from a single colony to ensure uniformity of the culture. Sequencing...to a shared computer or server. This will ensure that future members of your lab can access and use the...depending on the number of features we need to verify. Determining which features to verify will depend on...important features that differentiate this plasmid from its predecessor. Important features may include...reaction, or about 250 amino acids worth, so make sure the feature you want to verify is within this range. Read...plasmids, such as special growth instructions (temperature, strain, media supplements), suggested sequencing...information in a binder or digital notebook and make sure you note the freezer location of every plasmid in...