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...the template. The opposite is true if the sense strand is nicked for template formation: template will...mutagenesis. Error-prone PCR: This method amplifies the wild-type template with a “sloppy” version of PCR that...synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is degraded...low primer-to-template ratio is used to promote annealing of one primer to each template. The PCR product...and easy technique that can be used to generate complex libraries of mutant plasmids ready for deep mutational...in protein activity. See Natarajan et al for an example of this. Plasmid library generation prior to one...). It uses a uracil labeled wild-type ssDNA as template for a site-directed mutagenesis PCR that uses ...

...DNA strands complementary and non-complementary to the gRNA, respectively. When complexed with a truncated...full-length gRNA (20nt complementarity to target site) or truncated gRNA (≤15nt complementarity to target site...with which it is complexed rather than the protein itself. When native Cas9 is complexed with a gRNA that...transcriptional activator to the Cas9-gRNA complex. When this complex is directed to a target upstream of the...bioengineering, as it has facilitated the development of a simple and scalable means of making targeted genetic edits... directed to virtually any genetic locus when complexed with an appropriately designed small RNA, or guide...conventionally contains a 20-nucleotide sequence that is complementary to the target site, or protospacer, in the genome...

...target in the HR template, you usually would need to make mutations of the HR template to avoid this donor... HR template an issue? A8: Yes. If you have intact protospacer+PAM sequence within the HR template, it...for example) on each colony in order to screen for positive ones.  -- OR -- If your HR template has ...4 of the Cong et al. (2013) Science paper. For example, if you want to use double nickase, you could express...want to use wildtype cas9 or double nickase). For example, if you want to target two genomic loci with wildtype...homologous recombination (HR) Q7: Can you summarize HR template vector design guidelines? A7: A few notes below...you should consider using ssDNA oligo as donor template, it's usually more efficient than plasmid donor...

... and implant. A steady stream of photons from the laser to the patch cable to the ferrule implant must...opsin through a small piece of fiber optic cable implanted into the animal’s skull (right). The fiber optic...skull using dental cement (a similar procedure as implanting a guide cannula). A fiber optic patch cable is...various steps required to produce your fiber optic implant. Companies such as Thor Labs and Precision Fiber.... First, the length of the fiber optic for the implant depends on the dorsal/ventral location of your ...that is emitted from the end of the fiber optic implant scatters a great deal once it enters tissue, placement...penetrate brain tissue a short distance so ideal implant placement should be about 0.4-0.5mm above the site...

...bind to - particularly when you start using complex samples such as whole cell lysate or tissue sections...antibody signal in the retinal sample, but little to no signal in the liver sample, which you do (C), so your...signal in samples that have had the target protein expression experimentally increased. The simplest version...require variable expression of your target across samples - it is hard to distinguish between specific, invariable...proteomic and transcriptomic datasets already exist.   Example: You want to use an antibody to examine the levels...there is a wealth of transcriptomic data from this sample type, so you use your bioinformatics skills (or...Removing (or reducing) the protein of interest in your sample, should lead to reduced or eliminated antibody ...

...wells with an experimental sample allows for detecting one protein from the sample. A direct ELISA has many...require scientists to detect proteins in their samples. One technique that relies heavily on antibodies...principle of an ELISA is that any antigen within a sample adheres to the well (either directly or by binding...antibodies coating the wells to capture antigen from a sample, whereas antigen is bound directly to the wells...protein you are interested in detecting in your sample. When an ELISA kit is ordered, the wells are typically...already coated with the antibody. Then, you add your sample to the wells, and the antibodies bind to your protein... used to detect a single protein within a mixed sample. However, in this case your protein of interest...

...tasks that can be accomplished quickly and things that will take longer. Completing the quick and useful...useful tasks. The quick accomplishments will likely energize you. For example, let's say you’re doing ...project they’re a part of, completing them will give me some sense of accomplishment. I will prioritize which...its utility) How much time it will take you to complete the task When picking which tasks to actually...will swiftly give you the wonderful feeling of accomplishment while the longer tasks will give you larger...larger payoffs later. Don’t be afraid to accomplish some quick but moderately useful tasks at the expense ...experiment. The first way, it will take you 3 days to complete the entire experiment, but you’ll have to spend...

...production site to its final destination. As an example, most secreted proteins are produced in the endoplasmid... the untagged, wild-type protein. You may, for example, validate that the fusion protein localizes properly...visualized with a fluorescently-tagged antibody. By employing “marker proteins” which are known to be a part...intracellular targets. Therefore, it can be beneficial to employ well-characterized marker proteins tagged with ...fusions to visualize subcellular structures. For example, you might use your fluoresent protein fused to...and potentially which other proteins it forms complexes with. These “colocalization” studies give insights...within the same subcellular structure or protein complex. By coexpressing both the marker protein and the...

...fairly simple hierarchy: class to type to subtype to variant. Despite the seemingly complicated steps ... as Cascade (CRISPR-associated complex for antiviral defense) complexes, while Class 2 use single protein...tools using a Cascade complex is tricky, as you need to ensure every unit of the complex expresses at similar...present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with a...primary effector complex but are defined by the accessory Cas proteins in the Cascade complex. Despite a tenuous...the Cas effector may have the same name but be completely different! They are merely linked by a similar...protein Cas effectors, like Cas9. The Cascade complex doesn’t usually carry out the function of the specific...

...to the template strand. Therefore, I suggest that picking a crRNA complementary to the template strand...homology arms (60-70 bp) is added to the complex. This complete transfection mix, i.e. csRNP plus ssODN... Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively than Cas9 complexed with sgRNA...using the csRNP method is as simple as doing routine PCR assays. Here is a simple to-do-list to follow when...immunoprecipitated - along with its associated protein complexes - from the cell-lysate using antibodies against...against the target protein. The captured protein complexes are then analysed by mass spectrometry to identify...tool for guide RNA and donor DNA design, and implemented our optimised protocols into a medium-throughput...

...selectable marker, and target gene, for example, are amplified with overlapping regions of homology and...standardized constructs and tools for the rational and simple shuffling of DNA regions. Golden Gate Assembly ...Assembly Tool which is freely available online. Its simple interface can guide you through both experimental...fragment sequences and the polymerase used in the amplification. This tool allows instant updates as fragments...DNA – perfect for transforma­­­tion or as a PCR template. Some of the applications of the NEBuilder HiFi...with multiple point mutations NEBuilder HiFi can simplify multi-position site-directed mutagenesis by assembling...With NEBuilder HiFi DNA assembly, it becomes as simple as mixing fragments together with linearized vector...

...of primer pairs that PCR amplify in some states but not others. Because samples of many registers will ...states in a sample; instead, it will suggest the predominant state of registers in a sample based on a ...methods do not. How might one implement RSMs? In our study (1), we implement RSMs in E. coli with two plasmids...they were added. Figure 1D below demonstrates an example of a register that enters distinct states when ...not be very informative – each primer pair will amplify even if its target states are only represented ... determine the fraction of registers that are amplified by a particular primer pair. Then, by leveraging...infer the predominant state of registers within a sample using a metric that we describe in the Materials...

...coregulators exist as multiprotein complexes, a member of a single coactivator complex can serve as a rate-limiting...conduit to control the actions of the whole complex.  For example, the phosphorylation status of SRC-3 defines...interact directly with NRs) exist in steady-state complexes with secondary coregulator (or co-coregulator)...co-coregulator) partners. The coregulator ‘core’ complex is composed of a tightly-bound invariant group of proteins... bound co-coregulators associate with the core complex in a dynamic, regulated manner. The fact that coregulators... coregulators belong to distinct complexes may explain how more than 300 different coactivator proteins...defines its association with other members of the complex, such as p300 and CBP histone acetyltransferases...

...could simply be the percentage of the total cells present in the gate.   Figure 1: Example of yes-no... control (left) or with the sample (right). Negative and positive samples are clearly distinguishable ...designed your first flow panel, you’ll load your samples into the cytometer and see one of two results for...protein, a yes-no plot is the best-case result. For example, you have introduced a GFP protein into your cells...somewhere between a 104–105 signal intensity. If your samples and controls were processed the same way, the GFP...Created with biorender.com. Pro tip! If your samples and control underwent different treatment processes...place your gate based on the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing...

... into Sample6 as graduate students in Dr. Jim Collin’s Lab in 2009. From 2009 to today, Sample6 has changed...leadership, and grown to 25 members.  What is Sample6? Sample6 is an enrichment-free bacterial pathogen detection...current VP of Business Development & Operations of Sample6, for advice on how to take an idea developed during...etc. What project in the Collins lab inspired Sample6? The initial work focused on using bacteriophages... the focus of the application was changed, but Sample6 still leverages the core engineering and scientific... embodied, and driving the enterprise value of Sample6.  “Concisely and effectively presenting the value...clearly communicate their idea at various levels of complexity and abstraction and convince people with diverse...

...CRISPR/Cas9 ribonucleoprotein complex with linear DNA as repair templates which reduces time to generate...and the repair template of your choosing. Tools such as SapTrap Builder greatly simplifies design of these...cassette (Dickinson et al., 2015) made this step as simple as heat-shocking to induce the expression of a ...following a report that one can use linear repair templates (ie. PCR-derived dsDNA) with 30-60 basepair homology...worms are perfectly viable at 15 ºC, yet exhibit complete embryonic lethality at 25 ºC. This method resulted...optimize a range of editing parameters: oligo repair templates with homology arms of 35-80 bp, and DNA double-strand...CRISPR whether it be by improved gRNA or repair template design, delivery, screening, and more. Here are...

... to provide that context yourself. Simple words are not so simple Just as our lab used jargon that required... Addgene move from completing 2-3 large projects a year, on average, to completing 8-10 projects per year... team communication. Although these real world examples did not happen in the lab, they have parallels...problematic word. How many different things do you simply refer to as a “tube”? We took steps towards solving... that nearly all Addgene teams used that seemed simple, but they could in fact be interpreted in several... the aforementioned lab manager, said the word “sample,” did the word convey the same concept to Morgan... an Addgene-specific definition. Words such as “sample,” “material,” “item,” and “format” all received...

...the laser beam. A is simply the area under the resulting curve. By default, a sample on the flow cytometer...individual experiment. Practical example: lymphocytes in a mouse tumor sample You made it! So much about the...When using flow cytometry to analyze your samples, it is necessary to set up a sequence of gates to be...working with a heterogeneous cell population, for example from a processed piece of tissue, where cells are...size, while SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind...on the cell’s size, morphology, and structural complexity.   Height, Width, and Area Within FSC and...dive into the praxis of gating strategies. As an example, we’ll use one of my previous experiments where...

...nucleotides (for example, Digoxigenin-labelled Uracil) that will bind to complementary mRNA within an embryo...transcription to create dsRNA. Students can produce DNA templates via PCR off of the same plasmid and use these ...WISH protocol involves preparation of the tissue sample, several washes, and temperature changes to visualize...specific antibodies that detect the mRNA-riboprobe complex are added to the tissue. The last step involves...cells that contain this mRNA-riboprobe-antibody complex and, as a result, these cells turn blue. Students...create synthetic riboprobes off of linearized DNA templates which they can easily generate off of a circular...organism of interest, use the RNA to create a complementary DNA (cDNA), and then use PCR and molecular cloning...

...biology are not so simple, and engineering complex cellular behaviors requires making complex manipulations...organization scheme for multi-part DNA assembly. Simply put, it assembles individual DNA components into...predictable behaviors. While a gene is the most obvious example of an individual part, this category also includes...begin with a promoter and end with a terminator. A simple TU could be comprised of just three parts: promoter...MoClo. By a similar principle, TUs can be further complexed into multi-TU constructs, known as multigene constructs...manipulations to cells. For example: in a cell there are proteins which interact with other proteins and with DNA...DNA, and these complexes regulate the transcription of specific genes. What if one of those genes coded...