...hand. Records of plasmid construction were often incomplete or nonexistent. In the 21st century, many molecular...molecular biologists didn’t know the complete sequences or properties of the DNA molecules they were using...what the user wants to do and make the path to accomplishing their goals as intuitive and painless as possible...refinements in response to customer feedback. An example of this approach is SnapGene’s algorithm for detecting...could annotate a feature inaccurately. The best example is fluorescent proteins, which often come in closely... Addgene Additional Resources on Addgene.org Example plasmid analysis powered by SnapGene Browse All...

...not powerful enough for large brain samples. For large brain samples, we recommend pretreatment with ScaleCUBIC1...the stochastic expression of XFPs is possible by simply introducing a mixture of XFP genes. Stochastic ...time points, sacrifice the animals and fix the samples. When you use AAVs, it typically takes a few weeks...excessive amount of XFPs expressed. Then, clear the samples and visualize. For more details on this procedure..."Optical clearing and index matching of tissue samples for High-resolution fluorescence imaging using ...

...which has humanistic and philosophical implications. As an example, a group at Caltech used an optical fiber...and what it can enable? Boyden: There are many examples where you want to activate two sets of neurons...quarter of a million people have some kind of neural implant already. If we can use light to more specifically...Those are two of the biggest ones. And as a third example, it would be helpful to have molecules with better...three or four hour conversation. The brain is very complex and we've hosted hundreds of visitors in our lab...

... short science policy career and this seemingly simple question is a moving target. There are a multitude...pandemic has foisted upon us. Action Items:  A simple place to look for science policy folks is on Twitter... data gathering, regulatory efforts, policy implementation, program management, communications, diplomacy...setting: drafting legislation, making rules, and implementing the policy within the jurisdiction of the governing...working in science policy doesn’t have to be a complete career pivot. There are many ways to pursue your...proposals. Check out the Day One Project for some example proposals and consider applying for their accelerator...expertise and then once selected, you can pivot to a completely new field without issue. This may be more challenging...

... a photosensitizer composed of the protein complex complex and the MG-2I-dL5** fluorogen that can be used...thus forming the full FAP-TAP complex (MG-2I-dL5**).   A FAP-TAP complex will light up if excited with...-TAP MG-2I-dL5** MG-2I-dL5** is a new FAP-TAP complex where dL5** is the FAP. It’s job is to bind MG-...fluorogenic dye.  MG-2I is the TAP portion of the complex and is derived from the malachite green (MG) dye...

...non-trivial, however, as FPs are tremendously complicated entities with a large range of characteristics...about FPs than is currently represented at fpvis (complete spectral information, accession numbers, sequences...the goal of encouraging user contributions (using simple web forms) while also maintaining a relatively ...register with a valid email address).  One may also simply attach important references that serve to further...more powerful ways, and the advanced search page simplifies the process of building advanced multi-parametric... few years with an emission peak around 600nm?  Simply input these parameters and you’ll find matching...

...cell and any individual AAV particle is rare. But simply getting into the cell may not always be enough ...and therefore higher titer will be required. For example, retrograde AAV gets transported up the cell’s ...titers for successful transduction. In another example, neuronal gene expression by an anterograde AAV...visualize expression of fluorescent proteins, for example, as many viral particles must infect a cell to ... can optimize AAV production. We’ve listed some simple steps you can take below but you should read our...possible, use low-bind or siliconized tubes to store samples as it is more difficult for viral particles to ...

...started the process with, we got great amplification of our sample libraries. NEB also gave us a few goodies... aim to do large scale DNA preps with multiple samples without cross contamination. We currently use the...create a DNA library, one must first fragment a DNA sample into many small components. Without proper processing...repair Adapter Ligation Clean up/Size selection Amplification Clean up  I found this kit easy to use because... I am bombarded with a lot of information. For example, before going to my next workshop, I’ll ask myself...

...the requesters what they needed. In the example above, for example, we fostered a formal scientific collaboration...uploaded to the preprint repository BioRxiv. This example clearly illustrates the power of collaboration,...certain projects. The idea behind the platform was simple: any person working on COVID-19 could file a request...groups. In other cases, the collaborations were simpler and the volunteers helped with things like translations...need help with your scientific research. It’s completely free, of course. You can find more information...

.... 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...

...  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...

...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...

... 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 would be SNAP-...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...

... 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 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...

... 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...

... 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...