We narrowed to 888 results for: URE

...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 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...

...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...

...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...

...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...

...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...

...antibodies. Figure 1: Overview of using Sandwich ELISA to quantitatively measure protein concentration...format. Figure 2: Overview of using Competitive ELISA to quantitatively measure protein concentration...the plate. Figure 3: Overview of using Direct ELISA to quantitatively measure protein concentration...proteins. Figure 4: Overview of using Indirect ELISA to quantitatively measure protein concentration...competitive ELISA use antibodies coating the wells to capture antigen from a sample, whereas antigen is bound...coating the wells of a flat bottomed plate with a capture antibody that is specific to the protein you are...that also recognizes the same target protein. The capture antibody and the detection antibody recognize different...

...organism to target large or diffuse biological structures. Of primary interest here are systemic capsids...health. While it’s always helpful to start with a literature search to see what others have done, you’ll need... before using it in an application.     Figure 1: AAV toolkit for targeted gene delivery to the... antibodies developed in response to the first exposure to AAV9. The exception is the X1 capsid for targeting...strains. It is therefore important to review the literature for use of a capsid in your strain of interest...to start selecting and testing systemic capsids! Figure 2 contains a decision tree for selecting a capsid...further highlighted below the table.     Figure 2: This decision tree can help guide your choice...

...elevated temperatures. In both cases, GeoCas9 outperformed SpCas9 at high temperatures. See Figure 1 for ...stearothermophilus grows at temperatures ranging from 30-74 °C, with an optimal growth temperature of 55 °C. For a... longevity. GeoCas9 is active at high temperatures Figure 1: Thermostability of SpCas9 vs GeoCas9... These Cas9 proteins don’t work well at high temperatures, so to use them in thermophiles, bacteria must... to make eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally occurring Cas9 diversity via...CRAA (R = A or G) Spacer Length 20 nt 22 nt Temperature Range for Optimal Activity 35 - 45 °C 50 - 70...GeoCas9 RNPs were incubated in reaction buffer at temperatures ranging from 25-80 °C. Activity is expressed...

...publications really the best way to measure impact? As that Nature report also noted, papers detailing...scientific publications. Ventures like Impact Story seek to find ways to capture scholarly work that exists...scientific journals. Of course, no one measure will ever adequately capture a notion as complex as “scientific... you like to measure yours? More Reading Fenner M. "Altmetrics and Other Novel Measures for Scientific...Impact Measures" (2009) PLOS One 4(6): e6022. doi: 10.1371/journal.pone.0006022 Aragon AM. "A measure for...publish in journals with a high “impact factor,” a measure that dates back to 1975 and is based on the average...articles. Of course, a publication in Science or Nature is always nice, but in the wired world we are living...

...packaging as a single payload (Figure 4). Figure 4: ONE-GO biosensors measure the formation of Gα-GTP...uses new combinations of PE protein components (Figure 1), an altered epegRNA delivered with an enhanced...amplification of cargo, and heat treatment.  Figure 1: Left: recombined prime editing tools PE2max-...heritable desired edits in tomato and Arabidopsis. Nature Plants, 10(10), 1502–1513. doi: https://doi.org...N-terminal CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression...solution and when associated with microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule... inserted. B) Protein expression and labeling procedure. Adapted from Smith et al. 2024 under a CC-BY ...

...strategies such as the bisarsenical dye (Figure 1a) and FAP systems (Figure 1e) are inherently fluorogenic and...biological environments such as live cells and tissues (Figure 1). These flexible strategies give you the best...peptide tag could be used to label proteins in cells (Figure 1a). Other strategies that have been developed ...substrate ligand motif attached to a fluorophore (Figure 1b). Engineered ligases (e.g., lipoic acid ligase...attachment of a fluorophore ligand to a peptide tag (Figure 1c). Click chemistry (e.g., transcyclooctene–...amino acids can be incorporated into a protein structure and then used with the growing toolbox of bioorthogonal...fluorophore at the specific site of incorporation (Figure 1d). Fluorogen activating proteins (FAPs) – These...

...walks through the paper figure by figure. Some follow the same figure by figure organization with a different...adapt the second version (multiple presenter, figure by figure) if the primary purpose is training graduate...enjoy is discussion of current new and exciting literature. As I transitioned out of academia and away from...researchers of all varieties must stay current with literature and emerging technologies to remain competitive...in journal clubs. They also have likely had the pleasure of attending both productive and not so productive...pieces before that first meeting takes place to ensure success. One of the most important is letting people...ready for your first journal club to begin! Figure 1: Discussing next-generation sequencing at the...

... need to pick a fluorophore for each target and ensure the fluorophores, together, are able to give you...most efficient (i.e., happen most of the time). Figure 1 shows a graph from the BD® Spectrum Viewer, outlining...Fluorescence Resonance Energy Transfer (FRET).   Figure 1: Emission spectra and respective colors of a ... we can recognize a couple of fluorophores from Figure 1. For example, the chart tells us that PE and ...laser. To understand why that is, let's look at Figure 2. The blue laser excites at a wavelength of 488...maximum of PE's excitation spectrum (dotted line, Figure 2). The yellow/green laser, however, excites at...relatively stronger — or brighter — signal.   Figure 2: Excitation and emission spectra for PE. The ...

...transcript by endogenous processing enzymes (Figure 1). To ensure this tRNA processing only occurs on the ...recommends both for use in western blots (Figure 3).      Figure 3: HeLa cells transduced with SARS-...improvements in performance.      Figure 4: Structure of Voltron or Voltron2 conjugated to a JaneliaFluor...reconstituted within the target cells (Figure 6).   Figure 6: Illustration of split fluorophore ...Zeocin®, or nuclear expression of BFP2.     Figure 1: CROPseq-multi uses two sgRNAs with internal ... that incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in...just begun with PE7, it will be exciting to see future studies enabled by this tool as well as additional...

...will cover: Miniature CRISPR-Cas systems SEND plasmids Prime editing improvements Miniature CRISPR-Cas...problem. A miniature CRISPR-Cas system for mammalian use Stanley Qi’s lab has developed a miniature Cas system...paper in Nature Biotechnology Find the Cas12f-GE plasmids Programmed genome editing by a miniature CRISPR-Cas12f... guide RNAs Yong-Sam Kim’s lab also created a miniature yet active Cas system starting with the same Un1Cas12f1...nuclease Quanjiang Ji’s lab describes an effective miniature Cas system that utilizes a Cas12f from a different...ribonucleoprotein, and AAV.  Read the paper in Nature Chemical Biology Find the AsCas12f1 plasmids   ...paper in Science Find the SEND plasmids    Figure 1: SEND combines an endogenous Gag homolog, cargo...