...), which allows for the following to occur: A) Multiple polyclonal secondary IgGs can bind to different...sites per antibody. C) IgG’s are big enough that multiple labels (green circles in figure 2 below) can be...incubated together without forming large oligomeric complexes. Antibodies are also too large to be expressed...Antibodies Bivalent Polyclonal Can be labeled with multiple dyes Commercially available Not recombinant...sacrifice Good tissue penetration Can be labeled with multiple dyes Greater resolution due to lower label displacement...strength to a standard IgG secondary antibody (Pleiner et al figures 4A-C). And since they have limited...for colocalization labeling of up to 3 targets (Pleiner et al figure 4D). Unlike large secondary antibodies...

...BioRender.com.   Opto-G-protein coupled receptors  G-protein coupled receptors (GPCR) regulate cellular...consider when choosing one.     Figure 1: Example of an experimental optogenetic setup. A light source...light-gated ion channels, optogenetic G-protein coupled receptors (OptoXRs), and photoswitches. Light-gated...within a narrower band of wavelengths to allow multiple tools, such as biosensors, to be used in tandem...cellular function through multiple intra-cellular signaling cascades. Optical methods for controlling GPCR...than traditional pharmacology techniques allow. Multiple photoswitches can be used in a single pathway ...awry in a disease state.      Figure 4: Example of an intracellular signaling pathway activated...

... type of multimer present in your sample. In these instances, please note that the supercoiled dimer band...plasmid monomer from a plasmid sample, or if no monomers exist in your sample, you could try linearizing ...known as multimers, and they are the result of multiple plasmid copies combining in a process known as...control when performing a diagnostic digest is a simple but effective method for detecting plasmid multimers...Bedbrook & Ausubel, 1976). In fact, if your plasmid sample has a mix of different-sized multimers, you may...agarose gel). If the presence of a multimer in your sample remains unclear after visualizing it on a gel, ...consistent with the size of a supercoiled plasmid dimer. Please note that it is common to see other plasmid confirmations...

... in-frame protein fusions, and multiple cloning sites. In the simplest case we have a standard backbone... that consistently receives around 200 new DNA samples every week. Here we will provide an inside look...own plasmids. Staying organized With hundreds of samples coming through our doors, the first step to ensuring...our deposit process, and then we carefully track samples through our lab using barcoded tubes and plates...growth instructions (temperature, strain, media supplements), suggested sequencing primers, or mammalian ...assemble your expected full plasmid sequences (examples include Benchling, Serial Cloner, and Snapgene...process. Always sequence verify Once the physical sample and its associated information are at Addgene, ...

...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...afforded by coactivator complexes suggests that coactivators may be integrators of multiple cell signaling systems...datasets, for example, document the expression patterns of nuclear receptors across multiple different tissues...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...

...result is a heteroduplex, with overlapping strands of edited and unedited DNA. The heteroduplex is resolved...any base-to-base conversion in principle, but some changes require multiple steps and enzymes. Prime editing...repair allow for correct heteroduplex resolution? The prime editing heteroduplex is likely recognized as...CRISPR guide. Some examples include: TwinPE: two prime editors make complementary edits to opposite strands...editing is also more precise. Base editors, for example, typically edit all the target bases within the...edited sequence on the other strand. To guide heteroduplex resolution to favor the edit, the Liu lab turned...component of the MutSα–MutLα mismatch repair (MMR) complex, to temporarily inhibit this process and promote...

...scrambling. Everything we had set up involved multiple people in tight locations. Conference rooms for on-screen...talk to people and direct them. For interviews, being able to ask follow up questions, or ask people to rephrase...would have multiple interview subjects, I positioned a boom mic just out of frame allowing people to sit ...tough. Is the content accurate? Are we conveying complex ideas in the easiest way possible? Are the shots...we got sent home for what we thought would be a couple months. Jennifer and I spent about an hour that...home, how were we going to film anything? We had a couple options. The first was to put me on camera, filming...Magic Pocket Cinema Camera 4K. It’s wonderful and simple to use, but when you need to juggle filming and...

...control of multiple loci is deftly coordinated by the eukaryotic cell for the execution of many complex cellular...endonuclease activity coupled with a customizable single guide RNA (sgRNA) that bears complementarity to a 20bp target...Zalatan JG, et al.'s recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR ...toward tinkering with regulatory networks and multiplex gene control. However, these approaches are often...scaffolds to mimic natural transcriptional programs on multiple genes. Modifying the CRISPRi system for multidirectional...scaffold modules are comparatively small allowing multiple sgRNA constructs to be introduced simultaneously... utility of simultaneous and multidirectional multiplex control via scRNA design is readily apparent in...

...are prone to incomplete silencing of reprogramming transgenes, which leads to incomplete reprogramming... 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...vector-free cells Limited genomic integration Multiple transfections required Need to sequence to verify...-Free High High Transgene-free and vector-free Multiple transfections required Protein Delivery DNA-...can complicate comparisons made between lines. Incomplete silencing of transgenes is a concern as well,...

...generation simpler, as described in an earlier post on generating CRISPR mouse models. For simple knockout...challenging for sequences with complex secondary structure. Today, multiple companies offer long single-...length. There are plenty of Addgene plasmid options for templates - see the example at right using blue.... It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing typical...certain models remain more difficult to make. For example, large insertions, like a fluorescent protein knock-in...Quadros et al. recommend ribonucleoprotein (RNP) complexes of Cas9 protein and the separated guide RNA components...guide and your donor ssDNA. Here, we’ll use the example of inserting a 2A-FlpO cassette into the Fgf8 gene...

...of the promoter as part of a multiple cloning site (MCS). Having multiple sites allows you to easily orient...important that the digest go until completion. The time required for complete digestion varies for different...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...plasmid to the new fragment thereby generating a complete, circular plasmid that can be easily maintained... gene insert with respect to the promoter. For example, let’s say your plasmid backbone looks like the...    Alternatively, this whole process can be completed using a single enzyme if your insert is flanked...gel purification step, it is important to digest plenty of starting material. We recommend 1.5-2μg of insert...

... Many of these examples we’ve done annually - they are fan favorites! 6. Keep it simple This may be an...anything at all really! We've had plenty of events that included a simple corn hole set and a cooler. Chances...that requires people to be in groups, organize your troops prior to the event, and let people know exactly...power of simple word of mouth - be sure to bring up your event in casual conversation to get people excited...where there is food and beer. Ha! We had over 60 people join the fun - including a lot of future Addgenies...their teammates and project collaborators have completed their work thoroughly. It’s kind of a chicken ...gets much more manageable when you can enjoy the people you spend 40+ hours with each week. In conclusion...

...Zika and a closely-related virus, Dengue from multiple sample sources. SHERLOCK could also detect low-frequency... and/or are too expensive, time-consuming, and complex to use outside of standard laboratories. In the...protein variants, Cas13, and Cas12a, to develop simple, portable, and inexpensive platforms to reliably...with crRNA to target an ssRNA of interest, for example, a sequence specific to a virus or pathogen. Once...sensitivity of the assay, targeted DNA or RNA from a sample is first amplified using RPA (recombinase polymerase...reverse transcriptase (RT)-RPA, respectively. RPA is coupled with T7 transcription to convert amplified DNA ...quantitation without compromising sensitivity. To detect multiple targets in one reaction, Cas enzymes, such as ...

...template vectors, to perform multiple site mutagenesis and create complex fusion or recombination vectors...Combining multiple Level 2 vectors in the final assembly step permits the creation of more complex constructs...interest does x, but it also participates in y. Depletion of your protein may upregulate an alternative ... recently used by Stephen Royle’s lab, acutely depleted proteins from their compartment of action via ... Sci 2013 May 1;126(Pt 9):2102-13. Assemble multiple component plasmids from building blocks via Golden...Golden GATEway Cloning Frustrated by the complexity of assembling recombination of transgenesis constructs...cloning kit simplifies the cloning process for complex DNA constructs, particular for those involving ...

...Before digesting the DNA duplexes with Surveyor nuclease, save some of the duplex and run it as an undigested...products (BRAF-GFP duplex) and from GFP-edited amplification products (GFP-GFP duplex) as a control. ...Screening Using CRISPR/Cas9 Deliver Multiple gRNAs at Once - Multiplex Expression of gRNAs Generating Mouse...talks about. After months of troubleshooting, those people who did not tell you that essential thing ask incredulously...recommended by the original publication. Pro-Tip: 1. Use multiple gRNAs to target different regions of your gene...reasons to target different regions of the gene with multiple gRNAs, but the most important is that you could...target artifacts by comparing the phenotypes of multiple gRNAs. Targeting different regions of the gene...

... 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...words, or words that depend a lot on context. New people in the lab or collaborators will not have the context... 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...

...but what if we had a simple tool that allowed us to rapidly decode the complexity? The CombiGEM-CRISPR...individual (and multiple) genetic perturbations in single cells. Methods to scale up multiplexed CRISPR systems...was contributed by guest blogger Alan Wong. The complexity of biological systems can hinder our attempts...of providing an easy-to-use tool to analyze the complex combinatorial genetic networks underlying your ...experiments. CombiGEM-CRISPR: The marriage of two simple platforms CRISPR has revolutionized how we decode...mapping the combinatorial genetics that underlie complex regulation in biological systems. The CombiGEM ...products are a diverse pool of lentiviral plasmids. Multiple rounds of digestion and ligation result in plasmid...

...your sample to. For example, we use our negative control to compare exposure times of our samples. A prominent...Reproducibility  In order to attain non-bias assessment, multiple people in the lab conduct “independent screening”,...cannot be reproduced and projects that cannot be completed. A 2015 review analyzing and explaining the antibody...Baker, 2015), causing a huge waste of precious samples, time, and funds – up to US$350 million in 2015...techniques on rodent brains and is expanding to more complex brain systems such as non-human primates and humans...noise. We then compare the negative control to a sample with the primary included to confirm that the signal...antibody and not from the background noise. If the sample images similarly to the negative control, that ...

...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... protein of interest are pulled-down and later coupled to high-throughput sequencing to identify genome-wide...tool for guide RNA and donor DNA design, and implemented our optimised protocols into a medium-throughput...

..., we'll discuss the principles and process of designing a flow panel.  Principles of excitation and emission...Table 1, we can recognize a couple of fluorophores from Figure 1. For example, the chart tells us that PE...some background on the color spectrum and the principles of excitation and emission. The visible color...carry their emission peaks within their names, for example, BV421 and RB545. Fluorophores can also come in...en-us/resources/bd-spectrum-viewer.   Another example APC is a "bright" fluorophore when excited by the...building your first panel, we'll talk about one last principle of color emission. As you can see in Figure 1,... of neighboring colors can and do overlap. For example, APC overlaps noticeably with both BV605 and Alexa...