... (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...

...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...

...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 ...

... for something to brighten our day, whether it’s memories of summer or just a bright new plasmid. One ...identified from a high-throughput screen, selected for increased voltage response, brightness, and photostability...sensor dLight1.2 and voltage sensor Ace2N and additional GCaMP6 constructs: pAAV-CAG-dLight1.2 (AAV1)...One area with lots of bright new plasmids is our Fluorescent Biosensors Collection — scientists are always...iGluSnFR3 (Aggarwal et al. 2022). This new version has higher signal to noise ratio and is better localized for...variants optimized for different applications (v82 has higher affinity for glutamate but slower kinetics; v857... post. Figure 2: iGluSnFR3 displays enhanced brightness, expression, and sensitivity compared with earlier...

...relies on ultrasound waves as opposed to light to produce higher resolution images in vivo. “One of the ... result, ultrasound enables higher resolution images at depths up to eight millimeters – not single cells...New York recognized their limitations for peering right through living animals to see their organs – a liver...iRFP713 and iRFP720, is that they absorb and emit light in the near-infrared portion of the electromagnetic... melanin pigments, which absorb the majority of light below 650 nanometers (nm), and the absorbance of...toolbox In the last year, Verkhusha added two far-red light photoactivatable (PA) near-infrared fluorescent ...their fluorescence upon illumination with far-red light. As the researchers described in Nature Communications...

...measured. Archon1 is  bright, exhibits good localization, has high signal:noise ratio, high sensitivity, responds...blue light (445nm). The discovery and applications of protein partners that bind to only the light- or ...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...colleagues turned to optogenetics. Through the use of light-sensitive protein domains, they have developed a...system to control CRISPR-Cas9 activity using blue light (Bubeck et al., 2018). They accomplished this by...inhibits CRISPR activity.   With the addition of blue light, the terminal helices of the LOV2 domain unfold,... variant of AcrIIA4.” CASANOVA responds to blue light quickly. Within minutes of inactivating the CASANOVA...

...unprecedentedly-high brightness, bfloGFPa1, and the other with extremely-dim brightness, bfloGFPc1. The...Koike-Yusa et al., Nat Biotech. 2013. Dec 23.   Highly bright and extremely dim GFPs The availability of the...to low-intensity blue light. This approach relies on the incorporation of light-oxygen-voltage (LOV) sensing...made available through Addgene and we want to highlight it for use in your research. Martínez-García... the three dimensional structure of GFP, its brighter mutant S65T-GFP, and the subsequent biochemical ...variety of GFPs with diversity in fluorescence brightness, intensity, excitation and emission spectra. ... MitoTimer reporter using a Tet-on system (pTRE-Tight-MitoTimer) to show its usefulness in cell culture...

...visible light scatter The optical system has both visible and fluorescent light sources. Visible light illuminates...addition to visible light, flow cytometers can have a variety of fluorescent light sources. When interrogated... number based on intensity. Higher intensity events are assigned to higher channels. In a typical assay...experiment. Single-stained samples must be as bright or brighter than any experimental sample. In addition...interrogated with various visible and fluorescent light sources that allow users to assess a cell’s size...and provides useful information about the cell. Light that continues in the same direction as it was initially...size of the cell; larger cells produce more FSC. Light that scatters sideways to the path that it was initially...

...external light activation, you can’t use fluorescence to monitor processes directly affected by light. Long-term...nano-lantern emitted yellow-green light and exhibited 3-5 fold increased brightness over previous BRET probes,...Long-term light exposure can also lead to cellular phototoxicity, and experimental success can be affected...FRET, but it eliminates the need for an excitation light source. Instead, a luciferase substrate like coelenterazine...autooxidize and produce background fluorescence, so higher concentrations of the compound can be added to ... (eNL) described in Suzuki et al. improve the brightness of previous Nano-lanterns by 2-4 fold and permit...fluorescent imaging! These eNL constructs use the brightest characterized luciferase, NanoLuc, and its substrate...

...diagnosis. IDT, 2014. Dwight Z; Palais R.; Wittwer CT. uMELT: prediction of high-resolution melting curves...proficiency with common applications - now let’s dive right in! You know qPCR is most commonly used to measure...statistical significance (Taylor, 2017). With the right conditions and parameters, some people find it can...on hairpin design of probes/primers often offer higher allelic specificity, due to the need for the target...work better for rapid-cycle PCR, such as the LightCycler, as it reduces the primer/probe actions to a ...particular has been reported to work well in the Lightcycler while being relatively easy to multiplex (Thellwell...Agarose gel or uMelt analysis dsDNA dye (Sybr)  High No No No Melt curve Hybridization Probes...

...Arch (green-light activated proton pump) and ChR2 (blue-light activated cation channel) light-activated ...near-complete desensitization in continuous light. ArcLight Q239 - A GEVI based on the fusion of the voltage...deactivated using yellow light (590nm). BLINK2 potassium channel - An optimized blue-light induced synthetic ... is activated by cyan light and exhibits an inactivation maximum of 590 nm light. Aurora and Phobos optogenetic...non-neural cells, ion-based communication happens at a slightly slower scale.  All living cells with a membrane...model (Chernet et al., 2016). There are many other light-based genetic tools for studying bioelectricity ...fluorescent protein. This GEVI features increased brightness and improved plasma membrane specificity. Stable...

...COVID-driven virtual drain. Audience needs high speed internet access. High probability of real-time technical...social media… how do you choose? Which way is the right way?  First of all, I want to put to rest a common... areas of any given country have equal access to high-speed internet. Before you begin any work, make ...communication that you enjoy and that plays at least slightly to your strengths. Otherwise, you’re going to ...Resource Requirements Pros Cons Video Medium to high Uses visuals, audios, and text. Incredibly immersive... writing can difficult.  Photography Medium (high for microscopy) Visually compelling. Easy to convey...to make things more accessible by including, highlighting, or omitting details. Digital or print distribution...

...investigating any biological process. High quality genome sequence A high-quality sequence assembly of the ... Developmental biology insights using lineage tracing in zebrafish To highlight some of the features that...Lineage tracing studies highlight how these advantages can translate into insights in developmental biology...as a useful vertebrate model based on their straightforward husbandry and ability to produce abundant, ... a few of the available genetic resources and highlight two recent breakthroughs in developmental biology...lineage tracing approaches that led to important insights in developmental biology. Microscopy-based lineage... This microscopy-based lineage tracing defined a high-resolution map of early embryogenesis in time and...

...by an external light source, they have lower background signal and can be used in light-sensitive systems...Every few months, we highlight some of the new plasmids, antibodies, and viral preps in the repository...collection grows By Ashley Waldron We are excited to highlight the Anti-V5 tag antibody produced by our partners...constant domains, the plasmids for the heavy chain and light chain are also available from Addgene!   ...constructs to tag the GOLG-4 protein, mScarlet3 was 50% brighter than the original worm-optimized wrmScarlet. mScarlet-I3...to reduced background signal when tested in more highly autofluorescent tissues like the intestine. ...Expression of fluorophore-tagged GOLG-4 through eight stages of embryonic development. Representative ...

...500nm) Light 0.1 Crystal Violet Pre and Post Visible Light 100 Methylene Blue Post Visible Light 100 ... and Post Blue or UV Light 0.5 GelRed Pre and Post (Post recommended) UV Light 0.1 GelGreen Pre and...you to visualize your DNA using UV light – please note that UV light can damage DNA and that proper personal...are having trouble getting colonies, you might want to use higher competency cells. Additionally, if your...bacteria; add chemicals to precipitate out the high molecular weight genomic DNA; filter the remaining plasmid...100 Ethidium Bromide Pre and Post UV Light 0.5 For more information on these stains see the Bitesize...final product is going to be very large (>10kb) you might want to use electro-competent cells instead of the...

...editing rates as high as 83% with NHEJ and 40-50% with HDR. Coselection efficiency is so high that some applications...Every quarter we highlight a subset of the new plasmids in the repository through our hot plasmids articles...27798609 Listen to our mCyRFP1 Podcast Segment New bright monomeric red fluorescent protein - mScarlet! The... new RFP variants for increased fluorescence, brightness, and proper maturation. Three new RFPs were isolated...toolbox Increasingly popular Optogenetics tools use light to alter cell physiology and molecular processes...protein is irreversibly cleaved when exposed to light. Robert Campbell’s lab illustrates the utility of...this caging technique to beautifully demonstrate light-dependent Gal4 transcriptional activation and Cre...

...from an mRNA that forms a pseudoknot. At high copy numbers, high levels of the lncRNA bind to RepZ mRNA ... termination. These types of plasmids tend to be high copy number. Here, we will consider the replication... ori called DnaA boxes. These boxes come in both high- and low-affinity versions, but binding of the low-affinity...: it bends the DNA to redistribute the DnaA from high-affinity boxes to low-affinity boxes, allowing lower... redistributes DnaA (red) from binding primarily high-affinity DnaA boxes (dark red) to also binding low-affinity...DnaA negatively regulates its own expression, so high levels of DnaA act as a feedback mechanism to reduce...stringently controlled plasmids, replication is tightly coupled to the bacterial host's cell cycle, maintaining...

... more carefully controlled. Regulated promoters might act only in certain tissues or at certain times ...them on or off at will with a chemical, heat, or light. In the cell, promoters themselves are controlled...of other promoter elements, and tend to be more tightly regulated. Common promoters for eukaryotes and ...Repressed by ethanol Full length version is strong with high expression. Truncated promoters are constitutive...promoter from maize ubiquitin gene Constitutive  Gives high expression in plants. H1 small RNA expression ...polymerase III RNA promoter Constitutive  May have slightly lower expression than U6. May have better expression...depending on its orientation to your gene. T7lac High levels of gene expression Promoter from T7 bacteriophage...

...species like C. elegans and Drosophila. Light inducible promoters Light is another way to activate gene expression...biology use light to regulate transcription. Red flame plasmid pDawn contains the blue-light sensing protein...switched from an OFF to an ON state, and how you might use these in your research. When you're done with...inducible promoters Chemical agents, temperature, and light are all examples of factors that can lead to the...protein YFI. When light is absent, YFI phosphorylates FixJ, which binds to the FixK2 promoter to induce... preventing expression of a reporter gene. When light is present, YFI is inactive, preventing repressor...transcription to take place. Addgene also has yellow flame light-regulated two component systems designed by the ...

... inducing the brightest fluorescence (Filonov et al., 2014). Broccoli is twice as bright as Spinach2 when...Mango fluoresces nearly as brightly as Spinach, but binds with much higher affinity to its cognate small... their high binding affinity to TO1-biotin. Two of these variants exhibit increased brightness compared...V., et al. "RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking...biologist’s kitchen lab - and scientists using aptamers might know this best, as most fluorescent aptamers are...are 10-100 base nucleic acid oligos that bind with high affinity to small molecules to induce their fluorescence... the first aptamer identified to bind DFHBI, is highly unstable in vivo due to poor folding and thermal...

... variant has slightly different activation/inactivation kinetics. For example, the light-gated cation ...exists in several states: closed/light receptive, open, closed/light insensitive. If you deliver a continuous...to “reset” and become receptive to light once again. Therefore, light must be delivered in pulses. An example...the optogenetics coin is materials science-based. Light is delivered to your opsin through a small piece... optic cable implanted into the animal’s skull (right). The fiber optic cable is threaded through—and ...then connected from laser to ferrule to deliver light pulses to the target brain region. Thankfully, due...stimulation (the z-axis bregma coordinate). Because the light that is emitted from the end of the fiber optic ...

...the blue light molecule much, much, much more light-sensitive. Then you can use dim blue light and red ...the discovery of a light-sensitive opsin from algae that is sensitive to red light instead of blue, which...to tinker with brain circuits using the power of light. In a recent article published in Nature Methods...electrical activity of neurons on or off using pulses of light. By activating a neuron or set of neurons, you can...that all of these molecules are sensitive to blue light. Many have tried to make green molecules, but then...red light together. Understanding the brain through optogenetics research Addgene: What are some of the ... deep part of the brain, making it sensitive to light. When they illuminated this region, the animals ...