... 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 ...

... 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...

...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 ...

...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...

...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 ...

... not only brilliantly bright, but it also withstands denaturing conditions, high temperatures, and other...microscopy as well as correlative light and electron microscopy and observed high fluorescence resilience to ...retains folding and fluorescence in high molarity GdnHCl, at high temperatures, and in the presence of...Every few months we highlight a subset of the new plasmids, antibodies, and viral preps in the repository...CRISPR-Csm doesn’t degrade proximal RNAs nor does it have high levels of off-target effects, both of happen with...of antibodies we produced at Addgene and, as you might suspect, it has been one of our most commonly requested...8R] as another option for targeting GFP. But why might you need a second GFP antibody?  Similarities:  ...

...Signal Strength Lower Higher Sensitivity Higher Lower Background Lower Higher Post-translational ...bioluminescent reporter. This beetle emits a yellow-green light with a peak emission at 560nm. Shortly after the... which ideally would have low background signal, high sensitivity and of course be quick, accurate and...nature of the chemical reaction compared to using a high-intensity laser to rapidly excite GFP. As a result..., chemiluminescent reporters are generally less bright than fluorescent proteins, but have the advantage...Fluorescence Source of Emitted Photons Chemical reaction High-energy photons Kinetics of Photon Generation Slower...Subcellular Imaging Improving Well-established High-throughput assays Improving Well-established ...

...References 1. Viswanathan, Sarada, et al. "High-performance probes for light and electron microscopy." Nature methods...neuronal circuits and dynamics. FPs are generally bright, stable and well tolerated by cells. They can be...intrinsically fluorescent and can be detected with highly epitope-specific, fluorescently-labeled antibodies... signals or using anti-FP antibodies. GFP emits light in only a single channel. In multi-color imaging...the CNS. GFP and other FPs have the tendency to brightly label somata but not neuronal processes. In contrast...contrast, smFPs can label fine neuronal structures with high fidelity and at lower concentrations than GFP. smFPs...of these fine structures through their multiple, high-affinity binding sites for primary antibodies which...

...release. How is this light-mediated? The researchers split the proteolytically active light chain of BoNT/B...and SspB) that undergo light-induced dimerization. Upon application of blue light, iLID undergoes a conformational...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...Listen to this episode of the Hot Plasmids podcast Light-mediated block of synaptic transmission Article ...podcast segment An optogenetic tool that allows light-mediated silencing of synapses was developed by ...conformational change allowing high affinity binding to SspB, brings together the split botulinum neurotoxin...created. The depositors demonstrated the utility of light-mediated suppression of synaptic transmission in...

...response and brightness. They also do not spectrally separate from green sensors and blue-light-activated...Physiol 2018. PubMed PMID: 30819783. Light up CreLite: a red-light inducible optogenetic tool for precision...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...fluorescent protein (mScarlet) with either a seven or eight amino acid linker between them. The Ace-mScarlet...potential, Ace absorbance increases and results in higher quenching of mScarlet emission. Figure 2... with existing green-fluorescent GEVIs and blue-light-sensitive channelrhodopsins. Find the Ace-mScarlet...optogenetics. Cre activity is controlled by red light by splitting Cre and fusing each half with PhyB ...

...of painful responses in the left and right paw due to blue light activation and CNO silencing. (D) Immunohistochemistry...been made possible due to the collaborative and highly interactive environment that Janelia offers,” says...imaged 14 days post-transfection, excited at 505 nm (right). Scalebar represents 100 µm. Image from Abhi Aggarwal...expressed in the human cell line, Hep3B. From left to right: Mitotracker Red, GFP for the biosensor, Hoechst...changes following opioid withdrawal, providing insight into new methods for treating patients in recovery...involved in mechanical allodynia, a condition where a light touch creates a painful sensation. The dorsal horn...role in perceiving touch inputs. In this system, light activates the neurons (via oChIEF) and a chemogenetic...

...them to interact by stimulation with blue light.  Similarly, light can also be used to control homo-oligomerization...seconds after a pulse of blue light, and is reversible upon termination of light stimulation. CRY2clust enables...faster assembly and disassembly dynamics, higher sensitivity to light, and better distribution of clusters ...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...oligomerization of target proteins in response to blue light. This tool, CRY2clust, was created by adding a 9...from Arabidopsis thaliana. When exposed to blue light, CRY2 undergoes a conformational change that permits...drawback of this method is the stray excitation of neighboring neurons in opsin dense regions. To overcome this...

...of the sensor, dLight1.1 and dLight1.2, exhibit high (submicromolar) DA affinity and high-magnitude responses...(Figure 2). The dLight1 dopamine sensor In 2018, Lin Tian’s lab has developed dLight1, a molecular tool... of DA release.  Using dLight1 sensors in experiments The Tian lab used dLight1 both in brain slices and...Feelings of delight associated with the brain’s reward circuits are generally linked to dopamine (DA)...Similar to calcium and glutamate sensors, the new dLight1 sensor is a single fluorescent protein-based sensor...learning and reward consumption in live mice Find dLight sensor here! The GRABDA dopamine sensor Yulong ...binding (Sun et al., 2018). These sensors also exhibit high magnitudes of fluorescent responses. Studies on ...

...to increase plasmid yield. Keep in mind that you might need to use multiple techniques for a particularly...additional steps are needed to optimize yield. You might want to include an extra wash step or use a different...recommends DH5𝝰! This strain consistently gives good, high quality preps due to its endA1 recA1 relA1 genotype...repeats. We use NEB Stable to transform and prep high quality DNA that contains these elements. Be aware...wash step to remove endonucleases (see figure at right for an example). Look out for carbohydrates Certain...purification use the cell’s resources to produce high levels of protein, not plasmid DNA. If you need ... increasing the culture volume, be aware that a higher volume of lysate passed over the binding matrix...

...Every quarter we highlight a subset of the new plasmids in the repository through our hot plasmids articles...articles. These brief articles highlight the main features and applications of a partiular plasmid or ...parallel analysis that successfully identified new, highly specific agonists for orphan GPCRs demonstrating...are found in one third of all cancers, including a high percentage (up to 95%) of pancreatic cancers. Ras...an additional 6 gRNAs per gene generated using slightly relaxed gRNA design parameters. To demonstrate...method in bacteria that enables rapid, automated, and high-throughput genome editing is multiplex automated...otherwise unmodified bacterial strains (5). MutL is highly conserved in distant relatives of E. coli so it...

...sometimes these fluorescent proteins might not localize to the right places, which can provide misleading...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...efficiency of tobacco etch virus protease (TEV), a highly sequence-specific protease used to cleave affinity...work for evolution of other target proteins and might be useful for your studies.  Find the plasmids to...including fusions with Histone 2A or 2B can result in bright targeted expression while minimizing non-specific...microtubules of mammalian cells, the author found that too high expression of these markers can lead to non-specific...biotin and biotin-4-fluorescein to be dissolved at a higher concentration, and results in uniform NB biotinylation...

...Addgene, we've set out to fill this gap by both highlighting that researchers do learn MANY transferable ...the lab and by offering advice on areas where you might need some help. Today in our transferable skills...strategy to fix the issue. I was problem solving!  You might think that being able to figure out what went wrong...skills” and look at how scientists can develop and highlight these valuable skills. Hopefully, having a better...everything or revealed another issue in the process, you might need to go back to the beginning, reassess the situation...is just a question you have about the world. You might do some background research to better understand...issue. Depending on the particular problem, you might even be expected to write up a report to communicate...

...publication highlights In the table below, you'll find a list of publications we've highlighted in this article...Publication Plasmids   Highlights Komor et al., 2016 BE1, BE2, BE3   BE3 displays highest editing efficiency...the lab created HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017...fold less off-target editing than BE3, with only a slight reduction in on-target editing efficiency. To further... positions 4-7. ABEs 6.3, 7.8, and 7.9 display slightly wider editing windows of position 4-9, although...positions A3-A4 and A8-A10 at NGG PAM and 4.2-fold higher editing efficiency at non-NGG PAM variants compared... window of only a few nucleotides, so they are tightly constrained by PAM availability in the genome. ...

...using a highly competent strain that you can use for long-term library storage. After overnight growth,...popular classes of phage proteins to study. These are highly useful for the biotech industry, which is in the...the plate so you can regrow the lifted colonies overnight), flip the membrane over and place it colony-side...tested, as with any protein purification, but overnight at 30°C is a good start. Prepare a lysis solution...repeated 5 times) works well. Leaving the membrane overnight in the freezer during one of these steps is also...with your cells of interest and growing them up overnight. Assuming they expressed well, the proteins encoded... it can really help to incubate the membranes overnight in 500 mM to 1 M NaCl in order to enhance folding...

...color: they strongly absorb visible light to give colors in ambient light. In contrast, the widely used green...protein (GFP) gives a bright green fluorescence when exposed to blue to ultraviolet light, whereas chromoproteins...chromoproteins absorb visible light and give off color in ambient light, it gives scientists the ability...cases, cells containing high-copy plasmid-borne chromoproteins lost color overnight in liquid culture, though...colorful corals, to name a few. What’s behind these sights are an abundance of biological molecules like chromoproteins...Cassiopea xamachana (Blanquet and Phelan, 1987). Eight years later, scientists isolated pink, purple, and... irradiation by reflecting visible and infrared light to dissipate excess energy at wavelengths of low...

...substantially brighter and more photostable than fluorescent proteins, providing a straightforward way to improve...refinements to these systems—smaller tags, brighter conjugates, higher fluorogenicity, photoactivation etc.—...fluorophores conjugated to molecular species with high affinity for endogenous molecular targets such as...selectivity of the fluorophore attachment, the brightness of the resulting conjugate, and the complexity...exhibits low fluorescence when free in solution, but high fluorescence upon binding to its cognate protein...due to its established chemistry, small size, brightness, and cell permeability. In past decades, further... the Alexa Fluor and ATTO dyes, with improved brightness, photostability, and spectral range. Nevertheless...

.... The iBAR method resulted in high quality data even when used at a high multiplicity of infection, because...also function in high salt conditions - up to 100 mM NaCl. These tTALE-FPs also have higher DNA binding affinity...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...next generation sequencing.  In order to avoid a high false-positive rate discovery rate, conventional...human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low number of cells... levels within the cell. When levels of Ca2+ are high in the cell, calcineurin dephosphorylates the protein... the bacterial βgal assay, and are not suited to high throughput screening, calcineurin dynamics measurements...

... Zdk1 binds LOV2 with high affinity in the dark but dissociate under blue light. This tool has now been...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...up here.   Listen this episode of Hot Plasmids! Light controls the activity of a microtubule-associated... lab at UCSF to modulate cellular dynamics with light by modifying EB1. EB1 normally sits on microtubules...microtubule growth can be switched off with blue light which causes LOV2 and Zdk1 to dissociate due to ...photodissociable EB1 could be stopped in areas with blue light - a finding which opens novel ways in manipulating...Scientists are always on the lookout for better, brighter fluorescent proteins (FP) with improved tissue...

...development. However, careers outside of the lab highlight “transferable” skills such as leadership, management... impact, or a journalist does not understand the high-level science discussed in an interview, your project...be taboo in the lab, multiple research studies highlight the importance of taking a break and incorporating...network Attend informational interviews to gain insight into the daily routine of the potential job Join...and research 2 – 3 potential professions will highlight which skills you should focus on improving and... skill sets are strengthened. This short list highlights the ubiquity of skills across professions within... Outreach activities (e.g. Volunteer at a local high school science class: Science Club for Girls) ...

...cell is artificially high for both plasmids, so each plasmid “thinks” it has a higher copy number than it... 101 post, we’ll cover why this happens, how it might affect your work, and how understanding it can be...inhibits replication when copy number in the cell is high, but allows replication when copy number in the ...replication machinery when plasmid copy number is high (del Solar et al., 1998).  In cases of compatible...plasmids, though the partitioning problem does occur in high copy plasmids (Diaz et al., 2015). Plasmids contain... plasmid pairs into the new daughter cells.  For high copy plasmids, incompatibility due to partitioning...incompatibility due to having the same replication machinery. High copy plasmids with the same centromere-like binding...