We narrowed to 801 results for: DES

...discussed methods. As described above, you design two primers, each containing the desired mutations, to amplify...make a point mutation, you design two oligonucleotide primers with the desired mutation, then use those ... figure above shows guidelines for primer design; a desired insertion should be present in both primers...vitro recombination. Across all modifications described in Trehan et al., REPLACR-mutagenesis’ median ...

...coveted plasmid with no substantial delays. Design, design, design Before diving into the experimental work...experimental work. Fragments generation After you’ve designed your cloning scheme, you’re now ready to generate... to use 1-2 ug of the vector for digestion. PCR Design primers that overlap at least 24 bp with the template...from a large genome, you can recheck your primer design or optimize your primers or PCR conditions. Purifying...second and more detailed look at your experimental design. Full lawn of cells. The antibiotic in your plate...complementary to standard sequencing primers. If not, design primers to sequence your insert. When you get your...

... model to help them construct “designer” channelrhodopsins with desired characteristics. The model was...Viviana Gradinaru’s lab used machine learning to design new channelrhodopsins that are more light sensitive...constrained and poorly understood. It’s hard to design new channelrhodopsins with high-throughput proteins...Using machine-learning to overcome channelrhodopsin design challenges To overcome these challenges, the Arnold...experiments. The sequences in this library were designed using structural information about parental channelrhodopsins...existing channelrhodopsins CoChR and/or ChR2(H134R). Designer channelrhodopsins paired with a systemic AAV allow...Find our chemogenetics and optogenetics science guides ...

...utilizing chemogenetics (Designer Receptors Exclusively Activated By Designer Drugs, DREADDs) and optogenetics...beg the question: is axonal transport desirable or not desirable? If diffuse gene expression is tolerated...examining the use of DREADDs (Designer Receptors Exclusively Activated By Designer Drugs) to determine the ...to their Biosafety Level-2 or 2+. Please Note: Despite the fact that AAVs themselves are not pathogenic... CNS tropism (4). If axonal transport is not desirable, transport can be reduced by injecting a smaller...miRNAs), short hairpin RNAs (shRNAs), antisense oligonucleotides (ASOs), zinc finger nucleases, and many others...

...Marraffini’s lab took advantage of this lethality to design the first synthetic bacterial CRISPR system in ...gRNA cassette for plasmid curing through "self-destruction" after induction with L-rhamnose and aTet. CRMAGE...citrea scarless editing plasmids Sheng Yang’s lab describes a two-plasmid system that combines recombineering...Streptomyces (Cobb et al., 2015). pCRISPomyces-1 includes Cas9, a tracrRNA, and a CRISPR array, while pCRISPomyces...editing efficiency, perhaps due to its condensed design. For both systems, custom spacers/gRNAs are easily...et al., 2013), the Qi lab employed a gRNA-based design more familiar to those using CRISPR in eukaryotes...plasmid tools that can be used in this way. As described in the above CRISPRi section, the Marraffini Lab...

...expression in the retina.  Four approaches for designing synthetic promoters  To build a library of synthetic...synthetic promoters, the Roska lab used four different design strategies: Strategy #1 (ProA): Create promoters...promoters with the help of epigenetic analysis and designed using stretches of DNA that were transcriptionally...mice. Figure 1: The four strategies used to design synthetic promoters. Legend: TSS: transcription...class of cells  Of the four different promoter designs, promoters from strategy #4 had the highest rate...here.  Applications for synthetic promoter AAVs Besides restricting AAV transgene expression to particular...research for gene therapy. The Roska lab hopes to design improved versions of their current promoters, as...

...that requires modest gene activation. Synergistic Activation Mediator (SAM) Description SAM uses specially...-component system dependent on gRNA design (SAM) or peptide design (SunTag). This streamlines its delivery...genes across diverse cell lines.   dCas9-VP64 Description CRISPR activation can occur through fusing dCas9...requires a relatively simple construct, it exhibits modest levels of gene activation, with some genes experiencing...VPR and SunTag) (Chavez et al., 2016). SunTag Description Rather than using a single copy of VP64 per each...expressed consistently throughout cells. VPR Description VPR fuses a tripartite complex with dCas9 to ...

...DREADDs DREADDs DREADDS, or designer receptors exclusively activated by designer drugs, are G protein-coupled...Relationship Studies for Designer Receptors Exclusively Activated by Designer Drugs. ACS Chemical Neuroscience...Chemogenetics has become a popular tool in the field as it provides a noninvasive method for manipulating cell activity...Roth's and Takafumi Minamimoto's lab called deschloroclozapine (DCZ) shows promise as a CNO alternative ... Gomez J, Ramirez M, Hu X, Galvan A, Basu J, Michaelides M, Sternson S (2019) Ultrapotent chemogenetics...Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice. Nature...

... human gut flora. Guides targeted essential and non-essential genes with two guides per gene. The library...expressing guides that target essential genes were less represented while bacteria containing guides targeting... Jason Peters, Oren Rosenberg, and Carol Gross provides a streamlined suite of tools for using CRISPRi...existing bacterial CRISPRi methods. First, its modular design allows for customization with strain-specific promoters...s take a look at the essential gene folA which encodes the target for the antibiotic trimethoprim. When... modular nature allows for the customization of guides, promoters, selection markers, and different versions...

...of screenshots to highlight changes to both the design and functionality of searches. A better way to ...posts, protocols, and more. Selecting crispr in Guides will return links including our CRISPR guide.  ... isn’t new to Addgene’s plasmid search. The new design of the plasmid search page moves the filters from...Lentiviral’ which eliminates most of the plasmids designed for other uses. I could look through these resulting... information about the subject, link to related guides and protocols, and provide easy to sort tables ...to the CRISPR Resources page: The page links to guides, our CRISPR eBook, plasmid kits, and curated pages...by a convenient table of plasmids specifically designed for this purpose: Still stumped? Ask an Addgenie...

...This post was contributed by Patrick Miller-Rhodes from the University of Rochester Medical Center.  ...plasmids for your experiment at Addgene! How to Design a BiFC Experiment Much like FRET, BiFC experiments...quantification (Kerppola, 2006, 2013). These are described in more detail below: Fusion protein construction...each PIP and empirically test which combination provides the best fluorescent signal. As for linker sequences...single FP. Your control construct may generate a modest level of fluorescence due to the random association...lentiviral expression. For more detail on how to design BiFC experiments, detailed troubleshooting tips...Many thanks to our guest blogger Patrick Miller-Rhodes from the University of Rochester Medical Center...

...harnessing the homology-directed repair pathway, designing the best donor DNA, and avoiding common mishaps...knock-in. Then choose which Cas enzyme to use and design a gRNA to where you would like to introduce the...sequence to introduce, which is done through the design and use of a donor DNA molecule. Your donor molecule...strand break by homologous recombination.    Designing donors for HDR The basis of the HDR pathway relies...used as well. Below are some considerations for designing donor DNA for successful HDR events. CRISPR cut...need to be used due to synthesis limits of oligonucleotides. These donors traditionally have larger homology...or part of the guide’s recognition sequence is destroyed via mutation. By introducing a mutation in either...

...episodic memory, a special type of memory which includes autobiographical events (see Jin & Maren, 2015...neurons make to other distant brain areas. It is desirable for tracing compounds to outline neurons in the...applying small electric currents via inserted electrodes) may be applied, but these methods can have a...Nevertheless, the use of these tracers over the past few decades was essential to build up our current understanding...connections (Lo & Anderson, 2011). In the past two decades, several virus species have been modified and used...single synaptic step, thus preventing further widespread expansion of RABV. This improvement resulted ...out our retrograde AAV blog post. An additional desirable property for network visualization would be the...

...and some of the most popular commercial strains designed to avoid them. Why do I need an expression strain...protease: Strains where this is completely deleted (designated lon or Δlon) similary reduce proteolysis of the...which can prevent the culture from reaching the desired density within a reasonable time-frame. For these...and pLysE—the difference being that the latter provides tighter control of basal expression. What if I...don't see protein overexpression? The strains described above should generate sufficient expression levels...you’ve tried a common strain and don’t get the desired level (or any) protein expression? Low expression...is accomplished by growing your culture to the desired density at 37°C and reducing the temperature or...

...subtilis and Pichia pastoris pCri also includes plasmids designed for protein expression in systems other...construct, most often in special E. coli strains designed for protein expression. Cultures are then lysed...plasmids are divided into two sets, each of which includes plasmids with different varieties of tags, both...other eukaryotes better than E. coli. Plasmids designed for these systems may include a signal peptide...can improve disulfide bond formation. pCri also includes a special feature for difficult-to-purify membrane...membrane proteins. pCri vector pCri-13a, designed for use in B. subtilis, contains the MISTIC (membrane-...

...protein products is desired, as multiple selection markers can ensure that all desired tags have been integrated...integrated. Simply design your amplification primers with the desired targeting homology—in frame, of ...These tagging vectors are based on previously described pFA6a-link vectors and include a Kan, SpHIS5, ...former lab at UNC Chapel Hill. Bähler et al.(2) describe a modular collection of plasmids for a wide variety...3xHA, 13xMyc, GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. ...

...see: Uhlen et al., 2016). In this post, we will describe the approaches that can be used to determine selectivity...intended application — here, flow cytometry — is the desired target, using one or more of the approaches outlined...interpreting your data therefore necessitates careful design of RNAi sequences, confirmation of RNA knockdown...the target at different levels, it is possible to design experiments where the performance of the antibody...We find that cell tracker dyes are helpful in designing such panels, so that pre-stained cell lines can...pass type-1 membrane protein), which has been designated CD365 in the most recent HLDA workshop (HLDA10...recognized by either antibody was not shared. Tables describe that the antibodies both recognized the target...

...the virus doesn’t completely disappear; it just hides in latently infected cells. To truly cure HIV-1,... dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 ...Why can’t the body mount an immune response to destroy these reservoirs? Essentially, the immune system...enhance transcriptional activation. Zhang et al. designed multiple gRNAs to target the 5’ long terminal ...prevent reinfection of those cells. Kaminski et al. designed gRNAs to target the HIV-1 5’ and 3’ LTRs and expressed...could be used to combat this problem, just as ART includes multiple drugs to lower the odds of developing...strategy may be a better option than direct cleavage. Despite the potential difficulties in translating these...

...dystrophin transcript. Unfortunately, the oligonucleotides only modestly improve muscle function, and they must...wasting leading to death at around 30 years of age. Despite the amount of research conducted on DMD, there ... over time, stem cell editing would be highly desirable. Should stem cell editing occur, the CRISPR components... for Duchenne muscular dystrophy. gRNAs can be designed to flank a single or multiple exons that, when...Short-term CRISPR expression would be especially desirable, as it would reduce the potential of off-target...require robust stem cell editing to maintain the desired phenotype. Precision genome editing faces the challenges...Covello G, Denti MA. Exon-skipping antisense oligonucleotides to correct missplicing in neurogenetic diseases...

...plasmid with its elements described below.   Vector Element Description Origin of Replication ...molecular biology lab will undoubtedly be asked to design, modify, or construct a plasmid. A plasmid is a...why each is important. Our “Plasmids 101” series designed to educate all levels of scientists and plasmid...basics and spend more time developing cleverly designed experiments and innovative solutions necessary...utilized in the lab are usually artificial and designed to introduce foreign DNA into another cell. Minimally...the lab. Addgene’s Molecular Biology Reference includes information about molecular cloning, how to choose...

...Image Search results includes images that can’t be reused without attribution, despite filtering for images...Initially I didn’t have access to professional-grade design software, like Adobe Illustrator, and I didn’t ...Googling and some trial and error, I found some free design software that let me create graphics that better...of Adobe Illustrator. There’s both a web and a desktop version of this software. Vectr has layers, which... this post. It’s open source and available for desktop use for Windows, Mac OS X, and Linux. Inkscape ...The web-based collection of over 20,000 icons is designed by professional medical illustrators with input...

...multiplexibility in various species and describe an assortment of applications. Despite the ubiquity of CRISPR/Cas ... store a record of invading phages but to also destroy the phage upon re-exposure (2, 3). More specifically...complex binds to the foreign DNA, a cut is made to destroy the invader. Wikipedia, accessed 25 November...engineering. The diversity of the CRISPR/Cas systems provides powerful lines of defense against invading phages...CRISPR/Cas inspired genome editing was initially described by Cong et al. and Mali et al. in January 2013...Nine months later, more than 1500 publications describe work to improve the tool’s specificity, orthogonality...

...assign four different barcodes to each of the gRNAs and to embed these barcodes within the gRNA sequences...lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized GFP ...method that makes use of guide RNAs with internal barcodes (iBARs) in their loop region. After infecting ...bioRxiv 2019. https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast...intracellular Ca2+ (bottom panels) equipped with destabilized GFP calcineurin reporter (blue) and stained ...

..., creative writing, designing and curating exhibitions, animation, graphic design, and more. I recall ... career in neuroscience—whatever that entails. Despite being engrossed by the theory and findings of brain... my curiosity did not ultimately translate to a desire to do the research itself. Several years of exclusively...more creative faculties into motion, such that a desire to once again write for fun eventually saw an English...experimental aspect of my thesis: a survey instrument designed to measure how individuals respond to issues of...SciComm Series: Cognitive LoadSummer SciComm Series: Modes of CommunicationIntro to Our Summer SciComm Series...

... researcher in Jacob Corn’s lab, describes how you can better design your donor DNA template to achieve...Detect Zika Virus The RNA-based toehold switches described in this post are engineered to bind RNA sequences...Boston University School of Medicine, this post describes a tool you can use to study basic virus biology...researcher in the Silver Lab at Harvard University, describes R-bodies, “force generating protein machines that... Connie Cepko’s Lab at Harvard Medical School describes how AAVs function and indicates important things...from the Jackson Laboratory for Genomic Medicine described Casilio, a system that uses modular RNA binding...

...for Gibson assembly. The steps following primer design and the PCR process itself are very similar to ...the downstream steps. For this example, we will describe how to copy a cDNA from one vector into a new ...YGOI) for ligation into a recipient plasmid. Designing primers for PCR based cloning     The basic...: Do not cut within your insert Are in the desired location in your recipient plasmid (usually in ...examine the DNA sequence that we want to amplify and design primers that will bind to and replicate it. The...ends of the ORF and how these are used for primer design: Because we are cloning an ORF, we want to clone...TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but we need to use the reverse complement...

... C-terminal dronpa fusions. With these various designs, Xin has been able to develop photoswitchable Intersectin... other researchers will be able to use similar designs to create a wide variety of enzymes and proteins...post. Fatima Enam: Biosensors for human milk oligosaccharides Fatima Enam is currently a graduate student...fluorescent protein-based biosensors for Human Milk Oligosaccharides (HMOs). HMOs are thought to play an important...nonspecific in the second. Fatima therefore set out to design and produce two biosensors that could quickly and... and reliably detect HMOs. Fatima’s first design is a whole cell biosensor with two components - an enzyme...recently presented her work at her home town in Bangladesh where it was well received. In addition to her...

...fluoresce: FlipCherry. FlipCherry provides a proof of concept that the designed used in FlipGFP can be applied...range and high brightness. The Shu Lab from UCSF designed an improved apoptosis reporter, ZipGFP, which ...prevent binding, the β strands in FlipGFP were redesigned so that they are parallel to each other and thus...fluorogenic reporter toolkit, the Shu Lab also designed a red fluorogenic protease reporter based on superfolder...PMCID: PMC2117903. To, Tsz-Leung, et al. "Rational design of a GFP-based fluorogenic caspase reporter for...Central PMCID: PMC5026494. Zhang, Qiang, et al. "Designing a green fluorogenic protease reporter by flipping...

... encoding multiple guides. While two guides require two arrays for Cas9, two guides can be streamlined...applications. This set of new anti-FLAG frankebodies includes mEGFP, mRuby2, iRFP713, SNAP-tag, and HaloTag ...target multiple genes within a single cassette provides increased simplicity for combinatorial genetic...cost-effective alternative to commercial ladders which also provides a useful yet low-pressure teaching opportunity...less than half the size of either Cas9 or Cas12a. Despite its small size, CasMINI is comparable in efficacy...

...up walls of text. Since our guides are heavily used, our graphic designer made most of the Antibody Guide... quickly." Final design (right). Immediate and immense regret of the graphic designer not shown.   ...blog, website, social media, videos, newsletters, guides, and e-books – comes into play. Our goal is to ...communication between our content team and our graphic designer to make sure things are clear, accurate, and in...Reference image (left) provided to our graphic designer upon request that the science communicator "just...

...connecting each genotype with vulnerabilities) to design a therapeutic strategy that works. But, those darn...information on each cancer clinical sequencing report includes rare variants that haven’t been characterized?...based on maps of protein structure. Sometimes this provides a great deal of clarity. But, often these methods...signatures or cancer phenotypes. These assays were designed to complement the one assay per gene approach ...27478040. The lung cancer mutant collection is described in this paper. Systematic Functional Interrogation...the genetic pathway reference collections are described in this paper Systematic morphological profiling...  The genetic pathway reference collection is described in this publication We also wrote an extensive...

... Blog Learn Best Practices for Designing Your gRNA Read How Barcodes Can Be Used in Lineage Tracing Read...genetic perturbations. The ease with which one can design and synthesize CRISPR guide RNAs (gRNAs) for genome...their functions. The continual advancements in gRNA design necessary to achieve maximal on-target and minimal...regulation in biological systems. The CombiGEM platform provides a means to create barcoded gRNA libraries that...quantitatively analyzed by sequencing its set of barcodes. CombiGEM is highly flexible and can accommodate...PMC4151723. 2. Doench, John G., et al. "Optimized sgRNA design to maximize activity and minimize off-target effects...

...sometimes have to design new primers to sequence the important features. You can use primer design software such...such as Primer3 to help you design your custom primers. Remember, when designing a sequencing primer, you... to differ between plasmids, and selecting or designing a primer specifically to target this region, as...instructions on other factors to consider when designing your primers can be found here. Finally, you will... to Verify Your Plasmid Learn How Addgene Uses Barcodes to Track Samples Resources on the Addgene Website...

...engineering, with the ultimate objective of genome design.” It’s genetic engineering on a genomic scale, ...Systems Biology in November, Voigt and Alex Nielsen describe what’s possible when multi-input CRISPR/Cas genetic...with many guide RNAs connected because the signal degrades at each layer. If we could fix these things, then...many deposits to Addgene over the years. Can you describe the toolbox you are putting together?  Voigt: ...projects. Not from the ground up, but so that genetic designs of that scale could be created. Someone could take...doing is developing software to allow people to design systems of this scale. That will be coming out ...next 100 years, we are really going to be able to design new materials and chemicals as sophisticated as...

...application. It typically includes overview information, key dates, funding description, research plan, award... setbacks, and potential alternative routes, Provides opportunities to convince others why a project...aspects are understood. While reading the funding description, pay attention to the keywords used and think...grant applications restrict the space available to describe the proposed work. Thus, every single sentence...literature and offer educated insight into what the desirable features of future technologies should offer in...project are clearly stated and align with the description of the RFA and funding institution. Emphasizing...

...Resonance Energy Transfer (FRET) was originally described by Theodor Förster in 1948 as a variation of the... observed light emission by fluorescence. The widespread use of FRET with fluorescent molecules, including...FRET probe, try searching PubMed for articles describing the FRET tool that you are looking for and check...created the sensor that you need. FRET biosensors designed to measure specific small biomolecules or gene...biosensor, Cameleon (Miyawaki et al., 1997), was designed to measure intracellular calcium and published...1997. Since that time, numerous advances in probe design, fluorescent proteins and microscopy equipment ...molecules, detect cellular processes and signaling cascades, quantify mechanical tension (a molecular “spring...

...editing landscape. The need for PAM-less editing   Guides for CRISPR are typically selected by the ‘quality...sequence is suitable, but there is no PAM next to the desired cut site, then a further-away gRNA where there ... may wonder: how close is close enough for the desired edit to still occur if the cut site is sub-optimal...it depends, but generally the efficiency of the desired edit is reduced by over 50% when the cut site is...sequence, that of SpCas9, is NGG. Assuming all nucleotides are represented equally, that’s only a 1/16 chance... an ideal site: moving several bps away from a desired site does not have the same devastating impact ...Between SpRY and several of the other Cas variants described, nearly all genomic landscapes should be editable...

...Have no fear, Addgene is here to help!  General Design Figure 1: Two proteins are fused to the ...from the associated paper:   Color Base FP Description Article (Link to Plasmids) PI Blue EBFP2...contains both fragments of split mVenus (aa 155), includes I152L in N-terminal fragment An enhanced recombinant...Dan Mulvihill mVenus Split mVenus (aa 155), includes I152L in N-terminal fragment Constant rate of ...reversible complementation. TagBiFC (split HaloTag) provides maximum flexibility in color labeling with exogenous...single-molecule tracking in live cells.  Controls  When designing experiments, it’s important to include proper ...post for more on BiFC principles and experimental design. We hope you find these plasmids useful. Good luck...

...spreadsheet Data Field Description Plasmid Name Use a descriptive plasmid name and match the...lab. Plasmid Type Choose from: Encodes one insert, encodes gRNA/shRNA, or empty backbone. Purpose... in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options ...does this plasmid do?” in <200 characters. A descriptive purpose focuses on the practical application ... Primary Vector Type The vector type should describe how your plasmid is intended to be used. Choose...

...cassette via Cre recombination. Each knockout includes unique barcodes and universal priming sites to ensure ... the Chi-Bin Chien lab (Kwan et al., 2007) and includes four promoters, six fluorophores with nonoverlapping...The set of CyRFP constructs available at Addgene includes bacterial and mammalian mCyRFP1 or CyRFP1 expression...editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated with...codons) with kanamycin cassettes and were carefully designed to allow for easy removal of the resistance cassette...essential in B. subtilis 168. Each individual strain includes a xylose-inducible dCas9 paired with a single,...across a variety of plant species. This toolkit includes TALEN and CRISPR nucleases, nickases, and gene...

...BioMolecular Modeling and the CEO of 3-D Molecular Designs. Have you ever held your favorite protein in the...favorite protein in your hand? At 3D Molecular Designs, we create physical models of proteins using 3D...off a small, family-run business – 3D Molecular Designs – to commercialize some of the best instructional...reaction to this model on his blog. Or, for a description of how effective this model was in explaining... up close and personal, visit the 3D Molecular Designs web site. Or, if you are visiting a local classroom...check out our hands-on teaching tools that are designed to make the molecular world real for students....BioMolecular Modeling and the CEO of 3-D Molecular Designs.     Additional Resources on the Addgene Blog ...

...than plasmids without this DNA. They eventually designated this fragment as the ccdB region (for “coupled... serves as a counterselectable marker in the destination vector. Successful cloning occurs when the ccdB...restriction endonucleases in plasmid addition was first described by studies of EcoRI and Bsp6I in Escherichia coli...systems. As with the other toxin-antitoxin systems described above, one part of the pair is more stable than...toxin-antitoxin system discovered. Unlike the systems just described, the hok/sok systems consist of three components...long-term experiments. They’ve also helped researchers design and optimize cloning methodologies using counterselection...Central PMCID: PMC177048. Thisted, Thomas, and Kenn Gerdes. "Mechanism of post-segregational killing by the...

...unstructured templates and significantly reduced undesired edits (indels, etc.), while PE6c and PE6d were...Delivery to Intended REcipient Cells Through Envelope Design). This modular approach combines an interchangeable...Cell type-specific delivery by modular envelope design. Nature Communications, 14(1), 5141. https://doi.org...LOV-Turbo plasmids here! Next, Qin and Cheah et al. described TransitID, where two proximity labeling enzymes...that had been trafficked from the “source” to “destination”. Images adapted from Lee and Cheah et al. 2023...Figure 4: Time-resolved protein ticker tape. A) Design of iPAK4 protein ticker tape recording. B) HEK ...bioRxiv, under CC-BY-NC 4.0 license. This system provides a unique new way to record and quantify the dynamics...

... screen them for your desired edit. Good news! Remember the primers you designed to generate your pCAG-EGXXFP...CRISPR/Cas9 system floating around, and many pre-designed plasmids containing these variants ready for purchase...that do it for you. One such option is the CRISPR Design tool supplied by the Zhang lab. This simple website... and cons of using many different types of gRNA design software. When you are happy with your selection...Brighter fluorescence indicates a more successful gRNA design. Adapted from Mashiko et al., 2014. Not all...Resources on Addgene.org Find Software Tools to Help Design You gRNA Find CRISPR Plasmids for Use in Mammalian...

... your cells based on their size, the cytometer provides you with two measurements: forward scatter (FSC...SSC-H, or FSC-W/SSC-W, respectively. H, A, and W describe the shape of a histogram, which graphically represents... of the cell’s illumination (see Figure 2). H describes the maximum signal strength, while W results from...photocurrent vs. time. The resulting histograms is described through maximum current (H), the time the cell...depends on cell type and laser voltage. The voltage describes an electric potential that can be applied to the...comparing two markers simultaneously, we commonly describe the resulting populations as double positive (...that there are three parameters — H, W, and A — describing the intensity and duration of the detected signal...

...comment reflected the desire for a flexible generic solution, rather than an ELN designed for a specific research...to insufficient and non-standard record keeping despite scientist’s best efforts. We all have software ...practices, their opinions towards ELNs, and their desired functionalities i.e. main priorities of different...was a significant barrier to ELN adoption. This includes financial outlay, staff hours, troubleshooting... to require on-going maintenance and support.” Besides that, academic institutions tend to think about...required for regulatory purposes in many labs. Besides this being of crucial importance when applying ...

...any DNA sequence in the way that they like, the design and engineering of fusion proteins is relatively...fusion proteins and highlight some aspects of their design.  Protein size and shape matters The green fluorescent...necessary for post-translational modifications. Despite these potential issues, GFP has been used successfully...consist of random amino acid sequences or (ii) designed to form an inert, unstructured peptide and therefore...C-terminus of the fluorescent protein. I prefer to describe the different parts of the fusion from N- to C-terminus...2011).  Although structural information may guide design, it is advisable to generate multiple constructs...over-expression artifacts The introduction of DNA that encodes a fusion protein adds protein to the existing pool...

...optogenetics than neuronal activity. Researchers have designed a variety of photoswitchable elements into many... and limitations – for example, microtubule-destabilizing drugs are impossible to target to specific subcellular...plasmids if you want to make a stable cell line. This design allows highly localized, rapid and reversible microtubule...Rho GTPase activity. A) Schematic of Opto-RhoGEF design and activity. B) OptoTIAM (magenta) expressed in...processes in cells and tissues. And, this Opto-RhoGEF design could be extended to other GTPases. The Chow Lab...plasma membrane. In previous publications, they described tools to control formation of lamellipodia and...Jaber, Z., Gardner, K. H., & Chow, B. Y. (2022). Designing Single-Component Optogenetic Membrane Recruitment...

...your desired edit are all you need to generate a knock-in.   General considerations for designing a repair..., and ultraviolet light. In this post, we will describe the mechanism of homology directed repair, one...ssDNA templates (referred to as ssODNs (oligodeoxynucleotides)) are commonly used for smaller modifications...increase the frequency of edits, researchers have designed self-cleaving plasmids that liberate the targeting...information on HDR and CRISPR. Our CRISPR 101 series was designed to help explain the basic principles driving CRISPRs...et al. PLoS ONE 9(8); (2014). PubMed. Making designer mutants in model organisms. Peng, Y. et al. Development...

...optimized. How should I design my mutagenic primers? The key is to design primers that are the same ...generation of a plasmid library that expresses all desired variants of a protein. Applying a selective pressure...PCR that uses mutagenic primers to introduce all desired codon changes (step 1 in the image above). A second...ssDNA is packaged into phage and then extracted. Besides taking a couple days to generate the phage-derived... The one-pot saturation mutagenesis technique described in Wrenbeck et al is a PCR-based approach for ...Treating the plasmid with ExoIII and ExoI then fully degrades the nicked strand. Synthesize mutant strand: A...