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...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. ...

...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...

...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 ...

...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...

...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...

...Save time, go high throughput You are ready to perform your assemblies, but you have a high number to get...E. coli strains with higher transformation efficiency. If you’re cloning in high-throughput, we recommend...ligation efficiency, we recommend you calculate the right volume of each plasmid according to their concentration...time to think big and perform your assemblies in a high-throughput format. If your lab owns a liquid handling...restriction–ligation reactions in a 96-well (or higher!) format. Liquid handling robots are not only faster...experimental costs. If you are planning to perform a high number of assemblies, you will also likely need ...your bacterial stocks beforehand. You can also use high-throughput methods to streamline your plasmid extractions...

...amino acids (see example to the right). With such simple rules, you might think it’s easy to come up with... entire translation process. For example, during high growth rate conditions, E. coli preferentially upregulates...production of tRNAs that recognize codons found in highly expressed genes (Emilsson and Kurland, 1990). Controlling...produced at a lower rate than proteins encoded by highly abundant, charged tRNAs. For example Tuller et ... protein is encoded by a mixture of codons with highly and poorly charged tRNAs, different regions of ...usage, an organism can change which proteins are highly expressed and which are poorly expressed by producing... during amino acid starvation thus resulting in higher production of amino acid biosynthetic enzymes (...

...aggregation tendency, increase its brightness, and decrease the amount of light required for dimerization. She...Fluorescent Proteins Use Light to Control CRISPR Genome Editing Choose the Brightest Fluorescent Protein for...future award opportunities. Xin Zhou: Fluorescent Light-inducible Proteins (FLiPs) Fluorescent proteins ...create enzymes that can be turned on and off with light.  Xin hypothesized that she could make enzymes controllable...known to oligomerize when exposed to ultraviolet light. To do so, she generated fusion proteins in which...enzyme’s active site. In these fusions, ultraviolet light exposure caused oligomerization of the Dronpa domains...shutting off the enzyme. Later exposure to cyan light stopped Dronpa oligomerization and turned the enzymes...

...some light on multicolor imaging with fluorescent proteins. With the right microscope and the right choice...when choosing an individual fluorescent protein (brightness, photostability, and so on; see the previous ...colors and the mCherry spectrum is shown in the lighter colors; excitation spectra are blue and emission...this filter set, we would expect TagRFP to give a brighter signal than mCherry. In general, filter sets designed...supplement your cells with biliverdin for maximal brightness. In mammalian cells, one of the improved folding...fluorescent protein that is supposed to be extremely bright. In S. cerevisiae, we've tested a number of green...proteins with this filter set and have reported brightness measurements. Here, EGFP outperforms the improved...

...are commonly used in the lab as tools and we all might benefit from getting to know them a little better... injury, retroviruses generally have moderate to high immunogenicity, meaning they induce an immune response...transduce (infect) a wide range of cell types with a high efficiency. However, gamma-retrovirus infection ...tools have been derived from. Lentiviruses have slightly larger packaging capacities than gamma-retrovirus...immunogenicity, meaning it does not induce the same high immune response as retroviruses. This virus can ...been identified so far! Adenovirus generally has high immunogenicity, but some strains have been engineered... discussed in this article, adenovirus has the highest maximum titer, a plus for production and infection...

... university. My to-do list might look something like the image on the right (and for those of you who ...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...tasks at the expense of time spent on the long and highly useful tasks. The quick accomplishments will likely...you the energy to finish any additional work you might need to do. In most cases, I would recommend the...constantly switching between different projects, you might not be able to make any reasonable progress on any...to-do lists at the end of the day so they can jump straight into tasks the next day. If you work in a job ...

...ideal characteristics (low aggregation, high photostability, and high stability after fixation.) From the ...still being developed today. In this post, I will highlight the evolution of the  Brainbow multicolor labeling...zebrafish and Drosophila. Why Brainbow? Brainbow highlights the trend in biology towards single-cell resolution...development of a new technique. In a structure as tightly packed and interwoven as the brain, robust single-cell..., one would expect ten colors (see table to the right); in various reports, Sanes and Lichtman have observed...observed from 90-160 distinct colors due to higher copy number. The name Brainbow is truly fitting for...fluorescently distinct, their protein sequences are highly homologous, preventing design of antibodies specific...