...Protein Activation Library 113345 Activation Human Wright 3rd 7-14 58,570 Human miRNA CRISPR Knockout Library...

... Lin28A to somatic cells from separate plasmids Highly Efficient Reprogramming to Pluripotency and Directed...

...reagent you will be weighing out, a weighing boat or weighing paper to contain the reagent while on the...that you are weighing by looking for a weight range on the scale. Make sure that the weight of the material...out the material you are weighing onto the weighing boat or paper. If you weighed out too much material,... up. Discard the weighing boat or weighing paper in the trash if the material weighed is not biohazardous... Protocols Weighing Reagents Weighing Reagents Protocol Intro ... to handling the materials. Equipment Weighing boats or weighing paper Balance Scoopula or spatula Container...button. Before weighing out your reagents, determine the amount that you need to weigh. Gather the reagent...

...The route the light follows from the source to your eyes is called the light path . Light travels from ... Protocols Using a Light Microscope Using a Light Microscope Intro ... to encounter: the compound light microscope. As the name suggests, light microscopes take advantage of...compound light microscope with the main components labeled: Figure 1: Diagram of a compound light microscope...from the light source through a condenser that helps concentrate the light onto the sample, and then through...objective! Once on the higher objective, you will likely need to re-adjust lighting and focus. Increase ...basics of how to use a light microscope. Protocols...

...coronavirus using CRISPR-based SHERLOCK (Specific ​H​igh ​s​ensitivity ​E​nzymatic ​R​eporter un​LOCK​ing)... under either no excitation light, UV light or LED blue light.  Highlights: This is a one-pot, one-temperature...heat block, LED blue light illuminator or UV light illuminator. Approach: Highly similar to the SHERLOCK...captured by an antibody further along on the strip. Highlights: Of the 83 known COVID-positive samples tested... extraction but that may work only if there is a high enough (105 copies per microliter) viral load in...lateral-flow readout slip that allows rapid detection.  Highlights: Cas13a is not catalytically active in the presence... duplex and will not give a positive result for highly similar viruses. Unlike Cas12a, Cas13a does not...

...Microbial Opsins Opsins are light-gated ion channels or pumps that absorb light at specific wavelengths. ...respond to different wavelengths of light, like blue or yellow light. These various opsins also initiate...also a blue light activated cation channel. ChR2 is preferred over ChR1 because ChR2 has higher conductance...Channelrhodopsin from Volvox carteri 570 Chronos High-speed, light-sensitive channel from Stigeoclonium helveticum... 566 ArchT Light-activated outward proton pump from Halorubrum strain TP009. Improved light sensitivity...binding domain, allowing light to activate transcription. Phytochromes and light oxygen voltage (LOV) domains...) BLUF domains bPAC Light-activated adenylyl cyclase from Beggiatoa sp. for light-induced cAMP modulation...

...the Fab fragments contain highly variable regions, shown in light blue and light orange, that bind to a ...are light-sensitive molecules which emit a specific wavelength of light after excitation by light of a...pair the brightness of the fluorophore with the abundance of the antibody target. If a bright fluorophore...molecular tools used in the lab to specifically and tightly bind to other proteins. Antibodies, produced as...-efficient way to produce antibodies, with the highest variability in antibodies produced. Cells - Antibodies...protein chains: two large heavy chains and two small light chains. These four domains come together to form... to be produced in a single individual, giving a high probability that an antibody will be able to recognize...

...joining (NHEJ) pathway and the less efficient but high-fidelity homology directed repair (HDR) pathway....also enhanced specificity by engineering Cas9 into high fidelity Cas9s (hfCas9) . There is currently no ..., which are about the same size as SaCas9, with higher fidelity than SpCas9. FrCas9 (from Faecalibaculum... downstream of the target site (termed left and right homology arms). The length of each homology arm ...available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...strands Prime editing and base editing both offer high efficiency and low indel formation compared to HDR...large-scale, forward genetic screening. CRISPR can make highly specific, permanent genetic modifications to eliminate...

...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...

...These episomes can concatemerize, producing high molecular weight structures that are maintained extrachromosomally... a hybrid capsid derived from eight serotypes. AAV-DJ displays a higher transduction efficiency in vitro...separation of Rep and Cap shown in the figure on the right facilitates the viral pseudotyping discussed below... of transgenes, as well as the expression of neighboring genes. The short coding sequences of channelrhodopsins...any wild type serotype; in vivo , it displays very high infectivity across a broad range of cell types. ...

...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...

... (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...

...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...

... 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...

...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...

...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...

...out that with light-inducible photosensitizers and a quick zap of the proper color light, you can do just...2I-dL5**).   A FAP-TAP complex will light up if excited with light of specific wavelength and also produce...Publication Here Light-induced cell ablation with MG-2I-dL5** He et al tested out light-induced cell ablation...   Excitation with NIR light also produces less non-specific ROS because NIR light doesn’t excite natural...damage to neighboring cells. TL;DR MG-2I-dL5** is a NIR photosensitizer that allows for precise light-based...photosensitizer is a dye or protein that absorbs light to produce reactive oxygen species (ROS). The ROS...proteins or cells while having little effect on neighboring cells. The photosensitizer used in this work ...

.... Comparison to other high throughput cloning strategies There are several high-throughput cloning strategies...These enzymes might not, however, be appropriate for cloning genes from organisms with high CpG dinucleotide...High-throughput cloning, in a nutshell, is the systematic combination of different genetic sequences ...sequences into plasmid DNA. In high throughput cloning techniques, although the specific sequences of the genetic...biological fields. In synthetic biology for example, high-throughput cloning can be used to combine the functions...fluorescent reporters is a useful application of high-throughput cloning. MXS Chaining is one such technique...construct was flanked with a CMV promoter (to drive high-level expression) and a polyA signal (to terminate...

...positive population is no higher than 105 signal intensity. Signals any higher than this may not be clearly...to the right while decreasing the voltage will shift it to the left. If your signal is too high, lower...absence, the plots can look quite different. You might have high expression, low expression, and everything ...should be base expression — a peak to the right indicates a higher signal, or upregulation, and a peak to...average brightness from all cells that are positive for the marker of interest. MFI would be higher for a...GFP+ population cluster would be shifted to the right in comparison, say somewhere between a 104–105 signal...you can draw a gate which selects anything to the right of it, marking it as GFP+. Quantification for such...

...sequential administration (Chen et al., 2023). High viral titer High vector genome (vg) doses are recommended...systemic capsids are easy to produce in high titer, but higher doses (>1 x 1012 vg total in mice) may ...how systemic capsids are engineered, as well as highlight some considerations for their use. Then, we’ll... transduction efficiency in the brain along with high off-target expression, particularly in the liver...systemic capsid in question (or both). Therefore, a high-efficiency systemic capsid has better efficacy than... systemic capsids are… well, systemic, there’s a high likelihood of off-target expression, since they ...emerging capsids. Selected capsids are further highlighted below the table.     Figure 2: This ...