...insert expression (i.e., a strong promoter directs high expression, whereas weaker promoters can direct ...determines the type of plasmid and dictates how it might be used in the lab. Below are some common plasmid...particles, that can infect your target cells at a high efficiency. Addgene's expanding viral service offers...Next Generation Sequencing (NGS) comes in. NGS is a high-throughput, multi parallel sequencing platform that... Plasmids 101: 5 factors to help you choose the right cloning method Plasmids 101: Restriction Cloning...

...will have the highest specificity. To increase specificity, you can also consider using a high-fidelity Cas...gRNA targeting 5′ exon or essential protein domains High-fidelity Cas enzymes increase specificity. Dual-...a mammalian cell line that can be transfected at high efficiency Lentiviral transduction Cas9 and gRNA...

... capacity of >8 Kb. Constructs contain left and right arms to facilitate homologous recombination of the...and the adenoviral plasmid have matching left and right homology arms which facilitate homologous recombination...the shuttle vector, but this method results in a higher background of non-recombinant adenoviral plasmids...

...choosing the right cloning method for your research project . The following guide will highlight several of...

...from Addgene. 2nd Generation The graphic to the right shows how the lentiviral genome is edited down and...lentivirus is outlined in the simple schematic to the right. 3-4 plasmids are transfected into A293T cells: ...however, they are different viruses and thus use slightly different isoforms of these packaging components...

...immune system, antibodies are proteins capable of highly specific binding to other proteins. Antibody-based...technologies developed for optogenetics research utilize light to detect, measure, and control molecular signals...

...however, they are different viruses and thus use slightly different isoforms of these packaging components...research to reduce promoter interference and/or drive high expression of a transgene from an internal promoter...

...additional hypothesis-directed experiments. As you might have guessed from their name, pooled libraries are...

...polymerase) to bind to a promoter. Transcription is tightly controlled in both bacteria and eukaryotes. Promoters...

...limit of a light microscope. Due to the wave nature of light, an infinitely small point of light will blur... to provide high 3D spatial resolution while also avoiding the toxic effects of high light doses to the...fluorescence microscopy provides a high contrast image in which a bright signal overlays a perfectly black...placed to only allow light from the focal plane to reach the detector and all light from above or below...for example, red light instead of blue to excite a molecule of GFP. Because red light has approximately...approximately half the energy of blue light, two photons of red light are needed to excite GFP compared to only ...objective can a a high enough density of red-photons be establish for this to happen.Using red light has a second...

...UV-Violet Dark/Green N/A (high) High 503/ 518 Padron Monomer Blue Dark/ Green N/A (high) Low 503/ 518 rsCherry...specific wavelength of light. PA-FPs can be activated from low fluorescent states to high fluorescence states...fluorescence, to a brighter fluorescent state by exposure to a specific wavelength of light. The switch or...fluorescence when illuminated with 488nm light. However, when UV-violet light is presented first, it causes irreversible...illuminated with violet light, both variants are photoconverted to forms that emit brighter red fluorescence....when exposed to blue light, which is less phototoxic to live tissue than the UV light used by other PA-FPs...State Activating Light Pre/ Post Color Contrast (Fold Change in Fluorescence) Brightness Excitation/ Emission...

...with exquisite precision using only light. Optogenetics, or light activated tools, can control everything...an experimental optogenetic setup. A light source delivers light pulses to the subject/cells expressing... photoswitches. Light-gated ion channels directly open and close when exposed to light and change the ...changes with light stimulation to either activate or inhibit a specific receptor or pathway.  Light-gated ion...turn on with a light-pulse at one wavelength and stay on until a second pulse of light, with a different...pathways with light. This is achieved by coupling endogenous signaling molecules with light-activated partners... intensities of light sources used to prevent activation of both tools with each light source used.   ...

...dim" you might get the impression of a candlelight vs. a floodlight. For fluorophore brightness levels,...flow-cytometry-fluochrome-brightness-spillover Excitation Laser Color Very Bright Bright Moderate Dim...Photons of lower wavelengths are higher in energy, while photons of higher wavelengths are lower in energy...important to know the brightness of individual fluorophores. Table 1 describes the brightness of a few commonly...the cytometer. Brighter fluorophores have a stronger signal. Table 1: Degrees of brightness for a few commonly...-Cy7 are "very bright" when they are excited by the yellow/green laser but only "bright" for the blue ...leading to different levels of brightness, depending on the laser light's wavelength. Source: https://www.bdbiosciences.com...

... to their brightness and high degree of overlap. Dyes for DNA/RNA staining should have high affinity and...refers to the emission of light by a substance (fluorophore) that has absorbed light or electromagnetic radiation...whereby excitation of blue light will indicate where FITC is bound through green light emission. Key considerations...also vary in their brightness, depending on the respective fluorochrome. Brightness is impacted by both...of findings. If you’re struggling to select the right fluorescent tools, this blog is here to help. Here...ligand to make your selection easier, as well as highlight some recent advancements within this field. Fundamental...wavelength (Heinrichs, 2009). When a fluorophore absorbs light, electrons are elevated from a ground state to an...

...neuroscience. In fact, precise light-based control of biological processes is highly useful in other fields, ...consistent, and controllable pulses of light. Complicated patterns of light intensity and wavelength can be ...to low-intensity light, so off-target effects should be limited. Phytochromes are light-responsive systems...harness the power of light, Christopher Voigt’s lab created the first E. coli light-sensitive two-component...construct senses light, and an obligate chromophore allows the system to respond to various light inputs. In...various light inputs, and an obligate chromophore allows the system to respond. In far-red light or dark...exposure to red light rapidly deactivates the system. Version 1 of this far-red/red light-responsive system...

...LACE system for light-activated CRISPR-Cas9 effector, Polstein and Gersbach fused the light-inducible protein...binding affinity with light stimulation2. The Kuhlman lab created two improved Light Inducible Dimers (iLID...of Arch mutants screening for improved brightness. The brightest mutants were then further mutagenized ...enhanced sdChr construct, named CheRiff, exhibits high light sensitivity and fast kinetics. By combining both... weak or high activities (Bert, et al. 2000). To design pXPG, the authors incorporated a high-copy origin...repository, we can't give all of them attention they righlty deserve, but, in this post, we'll provide a small...2015 Feb 2;33(2):139-42. doi: 10.1038/nbt.3149. Light-inducible CRISPR-Cas9 system for endogenous gene...

...produce the same amount of light from an Fluc or RLuc reaction. In fact, NLuc is bright enough to detect endogenous...require external light sources. Unfortunately, detecting traditional luciferases’ dim light emissions requires...reporters produced bright signals without exogenous excitation and with OgNLuc being the brightest since it had...mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness and improved maturation time compared...Nano-Lanterns. RLuc8 is 5.5-fold brighter than RLuc but NLuc is 30-fold brighter than RLuc8 (6). Susuki, K, ... characteristics: No photoexcitation High Ion specificity High sensitivity Easy expression Gives ratiometric...coelenterzine substrate to the more stable yet brighter furimazine by Promega’s Chemistry group is a good...

...have higher specificity, lower cross-reactivity, and are more consistent lot-to-lot. The highest reproducibility... Tris-tricine 150 V, 1–3 hours Good for higher weight proteins Long running time   It’s important...during long-term storage and has both high binding capacity and high protein retention. It comes in a variety...usually preferred because of their higher specific activity, slightly lower cost, and smaller size. However...block with? It can be so overwhelming that you might just stick with the protocol your labmate handed...can help amplify a weak signal. However, they have high cross-reactivity, vary significantly from lot-to-lot... your assay. The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people...

...ambient light, and versatile as it can be activated by both low and high-intensity blue light. Unlike ...experiments that require high photostability, such as time-lapse imaging or with high light power detection of...Single-cell Phenotypic Observation and Tagging with Light (SPOTlight) as a way to isolate individual cells with...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...here.  Here's what you'll find in this post: Blue-light inducible Cre for use in bacteria CRISPR-Casɸ mGold...New items from the viral service A fast blue-light inducible Cre for precise control of bacterial gene... the use of optogenetics. Optogenetic tools use light to induce conformational changes in a protein.    ...

... FPs, which combine high cellular brightness and monomeric state, are highlighted in blue. aExtinction...consider effective brightness of NIR FPs in cells, in addition to their molecular brightness (a product of ...imaging NIR FPs, consider using light sources that produce enough light intensities in NIR. Usually, mercury...with blue-light controlled optogenetics, such as channelrhodopsins and tools based on light-oxygen-voltage...phytochrome (BphPs) photoreceptors have higher apparent cellular brightness than previously reported widely used...Molecular brightness vs. iRFP713, % Oligomeric state Photo-stabilityc, t1/2, s pKa Brightness in HeLa ...Baloban, M. et al. Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical...