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...for a given experiment. In the table below, we highlight the most common categories of genetic manipulations...joining (NHEJ) at the cut site Cas9 or Cas9 nickase High-fidelity Cas enzymes increase specificity Dual-nickase...editors Editing efficiency can be low, but generally higher than HDR pegRNA optimization can be challenging...location. Positioning your chosen Cas enzyme at the right spot within the genome relies on proper gRNA design...design a donor DNA to have the appropriate left and right homology arms. See Addgene's HDR blog post for more...guides that target closer to the edit site yield higher editing efficiencies. The template sequences can...specificity, consider using an engineered Cas enzyme with higher fidelity. In addition to off-target activity, it...

...episome which can concatemerize, producing high molecular weight structures that are maintained extrachromosomally...hybrid capsid derived from eight different serotypes. AAV-DJ displays a higher transduction efficiency in...on transgene expression and the expression of neighboring genes. rAAV episomes can develop chromatin-like...in vitro than any wild-type serotype, and a very high infectivity across a broad range of cell types in...Brain Connectivity Atlas and the Janelia Campus MouseLight project . rAAV vectors have also been developed...signals of cells (commonly neurons) in response to light. The short coding sequences of channelrhodopsins...wild-type). Another split AAV method which has shown higher efficiency in delivering large genes makes use ...

...opto-chemogenetic fusion proteins of a light-emitting luciferase and a light-sensing optogenetic element. When...neurotransmission. This, and the fact that GPCR ligands can have high potency, which is potentially important for dosing...off-target effects in vivo and the receptors exhibited high levels of constitutive activity, making them less...luciferin) is added, the luciferase enzyme generates light that activates the opsin. The opsin may excite or...illumination), making LMOs a versatile option. Coupling brighter bioluminescent modules, such as luciferase-fluorescent...example, a study in rats using an hM4Di receptor might consider an alternative DREADD ligand such as DCZ... chemogenetics by using physical and biological light sources for opsin activation. Proc Natl Acad Sci...

...), including overexpression plasmids with up to eight transcription units. Yarrowia lipolytica Golden ... Expression Tom Ellis Plasmids for constructing highly-tunable GPCR-based biosensors in yeast (requires...Bacterial Expression Naomi Nakayama A toolkit with both high cloning capacity and vector simplicity, compatible...in plants. The constructs are made with zCas9i, a high efficiency intron-optimized SpCas9-coding gene. ...TALEN Takashi Yamamoto Plasmids for construction of highly-active Platinum TALENs for use in a wide variety...

..., respectively). The bases are organized into a right-handed, double-stranded helix. To create the double... insert expression, as 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. Regardless of type, plasmids...particles) that can infect your target cells at a high efficiency. Viral Vectors , Viral Services Molecular...second-generation sequencing”) comes in. NGS is a high-throughput, multi-parallel sequencing platform that...

...on choosing the right cloning method for your research project . This guide highlights several of the ...

...for fast and efficient generation of large-scale, high-titer preps. This is thanks to established retroviral... on a case-by-case basis. Retroviruses carry a slightly elevated risk of integrating into proto-oncogenes...clinical-grade gamma-retroviral vector purification by high-speed centrifugation for CAR T cell manufacturing...

...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...

...polymerase) to bind to a promoter. Transcription is tightly controlled in both bacteria and eukaryotes by various... Cauliflower Mosaic Virus Ubi Constitutive Plant High-expression promoter from maize ubiquitin gene GDS...

...J., Bae, S., & Hur, J. K. (2021). CReVIS-Seq: A highly accurate and multiplexable method for genome-wide...Kochenderfer, J. N., Norberg, S. M., Hinrichs, C., Highfill, S. L., Somerville, R. P., Panch, S. R., Jin, ...

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

...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...

...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...

...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...

...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...

...Signal strength Lower Higher Sensitivity Higher Lower Background Lower Higher Photobleaching/phototoxicity...Luminescence Fluorescence Source of light Chemical reaction High-energy photons Kinetics of photon ...to be extra bright. Other luciferases have been modified to change the wavelength of light they emit or...bioluminescence is the firefly. Along with charming nighttime displays, these insects have contributed an important...reaction in its abdomen to produce yellow-green light (Figure 2). Despite the title of this post, this...includes all bioluminescence, is the production of light by a chemical reaction. In contrast, fluorescence...fluorescence occurs when a molecule is excited by a high-energy photon and re-emits a lower-energy photon; no chemical...

... (24-30°C). Brightness: Brightness is a measure of how bright is the emission. Brightness is calculated...is the emission of light by a substance that has absorbed light. The emitted light is at a longer wavelength...proteins are excited by UV light or blue light and their emission is green or red light. For example, T-Sapphire...lose the ability to emit light) after prolonged exposure to excitation light. Photostability can be as... & pHTomato (pH sensors), HyPer (H2O2 sensor), ArcLight (voltage sensor), and iGluSnFr (glutamate sensor...If you do not have a filter that will pass blue light to the detector/camera, then BFPs are of no use ...using more than one FP, make sure their emission light does not overlap in wavelength. In many microscopes...

...VVDfast homodimerizes under blue light iLID-SspB heterodimerizes under blue light BcLOV4 undergoes a conformational...membrane under blue light CRY2-CIB1 heterodimerizes or forms clusters under blue light Check out our Optogenetics...flip of a switch. You may be familiar with popular light-sensitive ion channels used to control activities...implant or a worm wiggling back and forth under blue light — but there’s a lot more to optogenetics than neuronal...into many other types of proteins. Here, we’ll highlight a few recently-deposited optogenetic tools to ...now, onto our optogenetic tools!  Opto-katanin: a light-powered microtubule-cutting sword Microtubules are...katanin) are recruited to microtubules under blue light, removing tubulin dimers from the microtubule body...

... need a higher antibody concentration to detect low levels of proteins. In this case, it might be faster...guide to good technique High background Antibody concentration too high Run a reagent gradient with...No signal variation in highly expressed proteins Antibody dilutions too high Run a protein gradient...Western blots can be tricky to get right. If you’ve thoughtfully done the technical design of your blot...still finding yourself having issues, you’re in the right place! In this post, we’ll talk about western blot...Figure 2: Reagent gradient. Here, the lanes are lightly marked on the membrane (1) using the gel as a guide...imaged. In this example, a 1:1,000 dilution gives a high signal with low background. Created with biorender.com...

...use a high fidelity ligase? Numerous applications have been developed to take advantage of the high fidelity...(i.e., are "higher fidelity") better than T4 DNA Ligase (21, 25, 26). Despite this higher fidelity, Taq...have a slightly lower Tm around the mismatch) over annealed fully base-paired substrates. Thus, high fidelity...for the high-throughput profiling of ligase fidelity that extends earlier studies through a high-sensitivity...researcher at New England Biolabs. When do you need a high fidelity ligase—and when is an alternative ligase...ligase from a thermostable bacterium that can survive high temperatures (up to 95 °C) and is active over a ...elevated temperatures (37–75 °C). T4 DNA ligase is highly active in nick ligation and DNA end-joining, but...

...REC lobe, light blue; bridge helix, purple; sgRNA, dark blue.   The REC lobe binds tightly to the repeat...yellow; REC lobe, light blue; bridge helix, purple; sgRNA, dark blue; target DNA strand, light gray; non-target... to produce SuperFi-Cas9, a high-fidelity variant that still retains high cleavage efficiency (Bravo et...microscopy structure (right, PDB ID 7S4X), viewed from the same angle to highlight conformational differences...yellow; REC lobe, light blue; bridge helix, purple; sgRNA, dark blue; target DNA strand, light gray; non-target... HNH, red; RuvC, orange; CTD, yellow; REC lobe, light blue; bridge helix, purple.   Apo Cas9 changes... ID 4ZT0), shown opaque (left) and transparent (right). The RNA guide sequence is extended as a single...

...away with slightly lower quality preps, while other applications require large amounts of high quality ...restriction digests. Transfection Grade DNA (high quality/high quantity) – Depending on the experiment, mammalian...viability. Avoid Low Quality/High Quantity – We recommend avoiding a High Quantity/Low Quality mindset... to use?  Obtaining higher quantities of DNA generally means starting with higher volumes of bacterial...to help you choose the plasmid prep kit that is right for you. What type of plasmid prep do you need? ...“low” is a relative term — even armed with the highest-competency cells you will still struggle to get...using is completely degraded. Cloning Grade DNA (high quality/low quantity) – You can usually complete...

...the bench overnight. Do not panic! Typically, antibodies are okay if they’re left out overnight, so mark...move forward:  Was a light-sensitive conjugate on the antibody left exposed to light?  Has this antibody...  First, fluorophores vary in brightness. You don’t want a super-bright fluorophore, like PE, on an abundant...Research the brightness of your fluorophores before buying and try to inversely pair brightness with abundance...storage, buffer considerations, and give just the lightest of nods towards conjugates.  Storage   Once you... compared to other data in a manner that demands high accuracy and consistency?  If the answer to any...chemiluminescent conjugate that can be activated to emit light with a simple kit, and is readily available on any...