We narrowed to 928 results for: mpl

.... HDR is crucial for certain applications, for example, correcting the allele that causes sickle cell ...manipulate that process. We began our research with a simple question: how do Cas9 and sgRNA interact with and...strand in Figure 1 below) was free to anneal to complementary ssDNA molecules in vitro. Figure 1: Cas9...discovered that single stranded donor DNA: Complementary to the released strand, 127bp in length, With...stranded donor DNA with other design parameters. Example single stranded donor DNA and a presumptive mechanism...target DNA that was accessible to anneal with complementary single stranded DNA. We thus wondered if such...

...to the Cas9/tracrRNA complex. D) The mature crRNA is used to guide the Cas9 complex to the target DNA which...requirements of these different systems can vary (for example, xCas9 is a variant with increased PAM flexibiliy...remainder of this post, we’ll refer to this RNA complex as the “gRNA”. The gRNA contains both the 20 nucleotide...using CRISPR/Cas9 for genome editing, researchers simply need to express a gRNA designed to direct Cas9 ...these arrays, they did not know their functions and simply called the repeated elements “direct repeats” and...

...the RNA from degradation, several simple methods have been employed which modify the sugar or phosphate... when scientists talk about their precious RNA samples. RNA is inherently less stable than most macromolecules...suggest coupling several of them together, for example phosphorothioates with 2’-O-methyl modifications...make your gRNA more efficient on a budget is to simply swap out several of the ribonucleotides for deoxynucleotides...RNA to control the entire editing system with a simple LED light. Lighting up your gRNA with fluorescent...

...  was used as a template for PCR amplification with a degenerate primer set to amplify IgG variable light... been thoroughly validated. The creativity in employing a variety of cloning techniques and troubleshooting...sequences. Fusion PCR was performed to create an amplicon that  was cloned into a plasmid backbone for expression... scientists working to visualize multiprotein complexes.  Fig. 2. Multiplex immunolabeling with...

... the antibodies time-sensitive, using precious samples, or being compared to other data in a manner that...conjugate that can be activated to emit light with a simple kit, and is readily available on any number of ...ain’t broke, don’t fix it. HRP is easy to find, simple to use, not sensitive to light in its inactive ...detected in the same assay. This is one of the most complex subjects in antibody-based protocols - but thankfully... this is an Antibody 101 post, so we'll keep it simple. If you follow the two basic guidelines below, ...compensation.    Working with antibodies can get a bit complicated, but hopefully this helps you successfully select...

...website navigation paths, the organization and completeness of our information, and the accuracy and relevance...relevant to a specific query and category. For example, a search scoped to plasmids shows filters for ...appear in our data into a single filter value. For example, there is only one viral prep filter for promoter...and whether it was a full or partial match. For example, matches of a name in the depositor field will ...specific catalog items as well as related items (for example, a kit that contains a plasmid with the catalog...engine unique. Most importantly, with this new implementation, we laid the foundations for many improvements...

... of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes, and...attached to self-labeling proteins. Some common examples of these self-labeling proteins are SNAP-tag® ...of protein substrates/peptides and antibodies. Example applications of this include coupling to an antibody...photons absorbed over the photons emitted. For example, DAPI is much dimmer than Alexa Fluor 488, and ...cells without causing undue toxicity. A prominent example is DAPI, a dye that binds specifically to the AT-rich...fluorescence-based techniques in biotechnology. Recent examples of advancements include the Janelia Fluor(R) (...finding the right dye for your experiment more complicated. We hope this post helps you in your search for...

...In complex metazoans, rapid cell division and large scale cell mobility are essential processes during... required for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...organism. Studying cell mobility within these complex environments has never been easy and, for many ...in thick specimens. See the video below for an example of two-photon intravital microscopy from PLoS Pathogens...scientists could easily track GFP expressing cells. For example, by harvesting lymph nodes at different times after...dynamics in lymphoid organs. In lymph nodes, for example, two-photon microscopy allowed scientists to better...time. Photoactivable fluorescent proteins, for example, have been successfully used to study B and T cell...

... on excitation power in a linear fashion. This implies that reducing the power 2-fold does not reduce ...fluorescent proteins can be directly compared. An example of a photostability measurement of various cyan...results in a loss of fluorescence, which, in its simplest form, can be described by a mono-exponential decay...intensity decay of a fluorophore does not follow a simple mono-exponential decay (Shaner et al, 2008; Bindels...applications in which far less power is used. For example you might find that you can get useful data from...fluorescent protein. In figure 1 we provide an example of such a comparison between cyan fluorescent proteins...

... The destination vector contains sites with complementary overhangs that direct assembly of the final ...plasmid (Option 1) or added using PCR-based amplification (Option 2). Advantages of Golden Gate cloning... reaction. For detailed Golden Gate protocols, complete with helpful tips and tricks, see The Sainsbury...is to "domesticate" your fragment: PCR-based amplification can be used to create silent point mutations...system is an incredibly powerful tool for cloning complicated constructs in a single, high-efficiency step....step. References: Sequential amplification of cloned DNA as tandem multimers using class-IIS restriction...

... or Cas nickase)  a guide RNA that targets the complex of TadA and dCas to the genomic DNA of interest...effect of removing the wild-type TadA from the complex the researchers developed a second set: The ABE8... the team replaced S. pyogenes Cas9 in the ABE complex with PAM-variant Cas9 proteins: the S. pyogenes... ability to use non-standard Cas9 in the ABE8 complexes enables a broader targeting scope for base editing... the corrected cells back to the patients. For example, defects in hemoglobin (𝛽-globin), which cause...that require high DNA editing specificity, for example for potential therapeutic approaches, the authors...

... features of the gene intact. Here, we show an example gene targeted for tagging with GFP at the C terminus...interest. For a C-terminal tag, as shown in the example above, we inserted a linker and GFP tag at the ...percent of cases, the stem cell will seek out a template for the repair process. By flooding the cell with...using the introduced donor plasmid as a repair template. This results in the insertion of the GFP tag ...tag precisely where we need it, in this example, at the C-terminus of the gene of interest. To get the molecular...CRISPR/Cas9 (Cas9 protein and the crRNA-tracrRNA complex) and the donor plasmid into the cells we use electroporation...

...plasmid are not always the best for others. For example, in our hands we find that a double harvest approach...larger genes such as Cas9. See figure 1 for an example of how changing transfection ratios can affect ... For some transgenes, transfection optimization simply is not enough to get a sufficient titer; this is... volume than with the unconcentrated prep. For example, at Addgene we typically concentrate 100-fold meaning...volumes of lentivirus routinely, you may opt for simply increasing the number of 10 cm2 dishes. While this...available, cost and downstream applications. For example, while the PEG precipitation method is relatively...

...the ribosome complex and Dr. Steitz quipped that RNA in the ribosome is in a complicated conformation ...Lowe lab have allowed them to begin understanding complex regulation of and by tRNAs and tRNA-derived RNAs...long hypothesized that life started with RNA and a simple cell membrane. His recent work shows that they ...replication with no proteins). The chemistry is complex and this work requires an interdisciplinary team... they do it. Of course, No conference would be complete without CRISPR Michael Terns from of the University...

...are often thought of as simple organisms , they have many levels of complexity, especially when trying...learning how to grow and care for the worms. As an example of one of the problems we work collaboratively ...We attempt to deconstruct and understand this complexity using existing C. elegans mutants. For instance...data is freely available, and the collection of samples and sequencing is being organized and funded by...Kittybiome project gives them access to rich and complex microbiome data for analysis. And, well, cats.....

...though three examples depend on many specific details about the individuals, each example provides a scenario...machine? Let’s walk through a more specific example. Perhaps you and your lab mate are negotiating ... something valuable. Importantly, as the third example shows, the ultimate resolution can involve ideas...your organization. In my non-academic roles, some examples of negotiation have been: A job offer conflicts...explore while also ensuring that my work gets completed? I want my company to pay for me to attend a conference...

...endogenously expressed SCF complex members, an E3 ubiquitin ligase complex. Finally, when auxin is added...fusion protein with the endogenous CRBN E3 ligase complex which will polyubiquitinate the fusion protein....target protein-of-interest and the CRBN E3 ligase complex leading to ubiquitination and proteasomal degradation...acid (IAA), the TIR1 F-box associated E3 ligase complex will ubiquitinate the AID-tagged protein which ...Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells. Proc...

...realized how perfectly flat the complex is on this surface”….where the complex is docked on the membrane of...like “Oh,…I didn’t quite appreciate that….” For example, when Tom Steitz was presented with a physical ... close to its reported cleavage site on the complementary DNA strand. And this is why we love working ...cleavage”. Jacob computationally added the non-template strand to the Jinek structure so that the resulting...double-stranded DNA, recognize a PAM sequence, allow the complementary base pairing between the guide RNA and the target...

... millions of people die from cancer-related complications each year. Despite these grim statistics, new...cancer, really? While cancer is extraordinarily complex at the molecular level, at the cellular level there...cancer research has focused on picking apart the complex cellular pathways involved in the development of...pathway. Its major appeal is that it enables the simple construction of vectors to study the function of...transcript most commonly found across all TCGA tumor samples is represented, making the results of any such ...biggest challenges facing researchers today is the complexity of the pathways involved in the development of...

...allowing very low background level of fluorescence. Examples for such photoactivatable or photoconvertible ... and their emission is green or red light. For example, T-Sapphire, LSSmOrange, and LSSmKate. Fluorescent...sensor), and iGluSnFr (glutamate sensor). More examples of these biosensors can be found at Addgene. ...also is also called Bimolecular Fluorescence Complementation (BiFC). 8 points to keep in mind when choosing...system can excite, and detect the emission. For example, if your microscope has only two lasers, at 488nm...minutes after it is translated to a few hours. For example, superfolder GFP (sfGFP) and mNeonGFP can fold ...