...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 ...

...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...

...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 ...