...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...

...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...

...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...

...of your employees every day. Prepare for a feedback session by writing down specific examples and notes...in. If a manager has concerns about an employee and the employee doesn’t know it, the problem is as much...with the employee. This is especially true if the problems are large enough that the employee should be...clearly and routinely. They are clear with each employee about what they do well and where they need to... clear about how the person is doing overall. Employees hate to wonder what you think of their work.  ...an unnecessarily big issue for you and for the employee. Make sure to focus on the action, not the person...Feedback will only have a positive outcome if the employee sees that real change is possible in your eyes...

...new assembly platform for generating both simple and complex DNA constructs Have you ever wished you ...Densmore’s lab at Boston University have greatly simplified the process of generating multiple E. coli gene...experiment, generate the necessary constructs, and complete screening in just 3 days? Thanks to Tyler Jacks...Sanger sequencing, respectively. The speed and simplicity of GMAP allows scientists to easily design, generate... generate, and test constructs composed of complex genetic elements. To demonstrate the usefulness of ...technique also allows for easy and rapid targeting of complex elements to specific genomic sites. For instance...the Toronto KnockOut (TKO) CRISPR Library. This complex second-generation CRISPR lentiviral library targets...

...from another within a fully stained sample. Figure 2 is an example of the emission spectra for two fluorophores...This complexity index ranges from 0-1, with a value of “0” indicating those markers are completely distinct...stained samples. If you are using conjugated dyes, it is critical that your controls and your sample antibodies...the primary detector for that fluorophore. For example, in figure 1, the FITC signal is spilling over ...cytometry relies on “unmixing.” Unmixing uses a complex mathematical algorithm and single stained reference...allows the user to use the autofluorescence in a sample as a fluorescent marker. Given that the level of... varies, it is critical to include an unstained sample for each tissue type you are running in your panel...

...other signal amplification techniques have been ruled out.  Ease of access Signal amplification isn’t the...approach is as follows: Prepare the sample. Incubate the sample with the primary (unconjugated) antibody... While secondary antibodies are useful signal amplifiers, the flexibility of their use varies across applications...conjugated in the lab. In the indirect approach, you can simply keep a stock of conjugated secondary antibodies...remove any unbound or excess antibody. Incubate the sample again with a conjugated secondary antibody, which...will need to be developed and optimized for the samples, antibodies, activation, and assay readout used... (And while I have it on good authority that a complete Western blot can be run in a single day, I really...

... conditions similar to humans. For example, due to their simplicity, yeast have become an important experimental...researchers develop a variety of therapies. For example, many scientists use mice to study diseases such...such as cancer since mice better recapitulate the complex interactions between cancer cells, therapeutic ... in other model organisms or cell culture. For example, Scott Lowe’s lab utilized mice to study how different...Yeast (Saccharomyces cerevisiae) Yeast, one of the simplest eukaryotic organism, is a commonly used model ...in breads and other baked goods! Yeast is cheap, simple and easy to work with as they can survive in a ...diseases that are caused by a specific gene. For example, several genes mutated in human cancers have a ...

... takes an hour or less to complete at 50 °C. After an hour or so, the sample is immediately ready to transform...to design primers for Gibson Assembly. For a simple example of using Gibson assembly, imagine that you ...will go over some advantages, disadvantages, and examples of how scientists are using Gibson assembly to...neighboring fragments can be created via PCR amplification with primers that contain the appropriate homologous... tools that help you design primers for PCR amplification of fragments to incorporate such regions of ...depicted below. First, you need to design primers to amplify the two fragments while also including regions ...vector or neighboring fragment. Then you would amplify the fragments and vector by PCR, verify that you...

...iPSC cell lines, which can complicate comparisons made between lines. Incomplete silencing of transgenes ...are prone to incomplete silencing of reprogramming transgenes, which leads to incomplete reprogramming...heterogeneity between clones. This can complicate comparisons made between samples, i.e. transgene expression and... of this method are that it’s relatively simple to implement and doesn’t require time-consuming production...pluripotency, but they also take less time to complete. Morphology is a simple and fast to evaluate. iPSCs should...generating iPSCs. The generation of iPSCs is relatively simple in concept: ectopically express a cocktail of stem...mutagenesis, the presence of a genetic scar, and incomplete silencing of transgenes. Overall, non-integrating...

...systems work using a multi-Cas complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade...compositions of the Cascade complex. Created with BioRender.com.   The Cascade complex Cascade is an important... Cascade complex from CRISPR type I-E. Created with BioRender.com. Thanks to this complex, developing...Cascade-Cas3 plasmids in Addgene's collection! A “complex” system Cas3 is part of the Class 1 CRISPR family... settings — mostly a consequence of Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR...component of the CRISPR Cas3 system. Most Cascade complexes are comprised of a combination of Cas5, Cas6, ...Cutting both strands ensures that there isn’t a template strand left behind that the cell can use to repair...