...modular cloning toolkit for genome editing in plants. BMC Plant Biol 20, 179 (2020). https://doi.org/10.1186...libraries depend on many gRNAs delivered with a relevant Cas nuclease. MoClo can be used to assemble multiplexed...are not available to those working with yeast or plants. Using MoClo, or better yet, a pre-made MoClo kit...achieve a desired effect. This is commonly used in plant biology to achieve traits such as disease resistance...blog on choosing a MoClo strategy and kit if you want to learn more about this cloning technique! Many...Cloning Guide EcoFlex MoClo Kit CIDAR MoClo Kit MoClo Plant Parts Kit Resources on the Addgene blog High Complexity...

...equipped using fast-acting optogenetic tools. The advantages provided by chemogenetics are instead that it...Targeted and specific delivery becomes especially important for studies in neuroscience where specific tissues...neuron need to be targeted. Targeting is also important in the development of therapies when expression...Picking Ligands Picking the right ligand is also important for building a chemogenetic system. Your experiment...cellular response you aim to manipulate. It is important to consider that the same receptor can elicit ...drug that only binds to one receptor, so it is important to fully characterize the compound, or compounds...will promote hyperpolarization. PSAM ligands The advantage of PSAMs lies in their ability to be specifically...

...process involves a significant amount of QC. First, we perform Picogreen quantification on each and every...high-quality isolated DNA samples of sufficient quantity for sequencing. This process is completed in a... preparation process and includes Picogreen quantification of various intermediate steps, a sizing electrophoretic...electrophoretic gel, and qPCR quantification of the final libraries. A broader view of the whole plasmid... Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral...

...selectable with the antibiotic whose resistance gene is encoded in the viral vector. This antibiotic selection ... 320,000 viruses can infect mammals. Even more abundant are the Earth’s estimated 1031 bacteriophages ...that infect bacteria), many of which are doing important work in our microbiomes. Given that viruses are...tips for your virus experiments. References 1. Anthony, Simon J., et al. "A strategy to estimate unknown...hepatitis B virus DNA and hepatitis C virus RNA quantification as measured with branched-DNA technology." ...PMC84922. 5. Ugai, Hideyo, et al. "Stability of a recombinant adenoviral vector: optimization of conditions...

...fragment. The availability of both wild-type and mutant loxP sites has allowed scientists to leverage this...site-specific recombination. The FLEx switch takes advantage of the orientation specificity of the site-specific...needs. How does the FLEx switch work? Let’s say you want to design a genetic FLEx switch that turns BFP expression...followed by the mCherry coding sequence in the antisense orientation (Figure 1, top). The entire DNA cassette...the gene you would like to rescue with in the antisense orientation (let’s call the gene “GeneR”). The...that your gene of interest starts in the inverse/antisense "off" position and is flipped to the sense (on...Saunders A, Johnson CA, Sabatini BL. 2012. Novel recombinant adeno-associated viruses for Cre activated and...

...smallest program. I don't mean have fancy food or a fantastic view (these are nice if you can afford them). ...performance with room for fortuitous improvisation. You want to make it look effortless to the audience, but ...so her ability to control the event is what is important. Choose 4 panelists (maybe 5) and no more. Too...on the topic at hand. You can use this to the advantage of the program. Start the program by polling the...Timing is everything Many of my suggestions are meant to keep the event on time. Nobody likes a program...postures shift, they are checking out. If you can, plant 1-2 good questions with trusted audience members...section. And what about those audience members that want to take over? It is the moderator's job to politely...

...first things you might want to know is what their project is. Or maybe you want to introduce yourself ...poster: those that want you to walk them through the entire poster, or those that want to look through themselves...to introduce yourself and what you do. You might want to include your university and your advisor’s name...like “Let me know if you have questions or if you want me to walk you through the poster.” Have a notebook...there. Check out the conference website for any relevant hashtags and handles to help you find other scientists...

...patients. And, to be honest, I’ve never enjoyed grant-writing. The more I talked to people with industry...interested in the drug discovery process, or you want to end up in an industry job, being immersed in ...on which company you choose. If publishing is important to you, make sure that your project will not involve...pages. In academia, however, writing papers and grants is an inescapable and essential part of being able...’re often required to at least attempt to write grants. If you don’t enjoy or excel at writing, an industry...postdoc will free you of the never-ending cycle of grant-writing that is academic life. Of course, because...contrast industry postdocs at those companies. Do you want a postdoc that’s shorter? Longer? Would you like...

...PEG precipitation of the HEK293 cell culture supernatant because it’s time consuming to prepare the 40%...virus from the cell pellet alone. If yield is important, or if the viral particles of the AAV serotype... concentrations of iodixanol to separate out contaminants from an impure AAV preparation. 15%, 25%, 40%...titration by qPCR Overview: AAV titering by qPCR quantifies the number of genome-containing viral particles... primer/probe set can be used and samples are quantified by comparing to an AAV standard curve, which ...Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived...R.A., & Samulski, R.J. (2007). Production of recombinant adeno-associated viral vectors for in vitro and...

...called Cascade (CRISPR-associated complex for antiviral defense). Cascade is brought to the target location..., with Cas3 systems making up type I, the most abundant type in the Class 1 category. These type Is can....com.   The Cascade complex Cascade is an important component of the CRISPR Cas3 system. Most Cascade...is pulled through the Cas3. This mechanism is important for Cas3’s biggest trick. Cas3 likes to bluff,... pairs, stopping either at boundaries defined by anti-CRISPR proteins or running into other proteins naturally...to its full potential. Hopefully more people will want to utilize Cas3’s degradative power in the future...CRISPR 101: Cas9 vs. The Other Cas(s) CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems INTEGRATE...

...would at least be guaranteed a successful result? While there are never any guarantees, some cloning techniques... purposes. We’re here to help with that! Four important factors to consider for any experiment are: ...Efficiency For cloning in particular, another important consideration is: The number of inserts ......Resources” section at the end of the post! Speed: Your grant deadline is approaching or your advisor is demanding...looking for a more streamlined approach, you might want to give Gateway a try. This method, developed by... to the use of toxic ccdB to prevent any non-recombinant vectors from propagating. The ligation-independent...

... is our own immune system, which can recognize antigens on cancerous cells and eliminate them from the...ensues, traditional chemotherapies have taken advantage of the universally-dividing behavior of cancer...approaching the Ras problem by exploiting the different mutant versions of Ras that are most prevalent in human...fluorescent protein fusion studies and the generation of mutant cell lines. Further, for each plasmid in the kit...making the results of any such study as clinically relevant as possible. Connecting the research community...community, will soon expand to include several other important pathways. We invite feedback and contributions...of signaling pathways with potential to confer anticancer drug resistance." Science signaling 7.357 (2014...

...of detection; they are useful almost anytime you want to express multiple genes in the same cell. Although...expressed from the same cassette is sometimes advantageous, particularly when only a portion of the plasmid...expression levels between two or more genes is important. How do multicistronic vectors work? Scientists... mRNA. However, some bicistronic vectors take advantage of an element called an Internal Ribosome Entry...bicistronic vectors; however, they do have some disadvantages. These elements are quite large (500-600 bp)...vector.3  2A Peptides To overcome some of the disadvantages of the IRES element, scientists have adapted...PubMed. 5. Scalable signaling mediated by T cell antigen receptor-CD3 ITAMs ensures effective negative selection...

...derived. For a breakdown of GFP, it's variants, and their relevant mutations, check out Marcy's post from...FPs just keeps getting larger with hundreds of variants available. Read on to familiarize yourself with...emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP (CFP), and yellow FP (YFP...calculated as the product of extinction coefficient and quantum yield of the protein, divided by 1000. In many ...temperature). pH Stability: This parameter is important if you are planning to express the FP in acidic...with very comprehensive data sheets on many FP variants Plasmids 101: Green Fluorescent Protein (GFP) ...

...it is limited to only 20 bases, or why it’s so important to choose a target sequence with a nearby protospacer-adjacent...unpaired strand in the center of the protein. The repeat:anti-repeat duplex pokes out from the REC lobe and...blue.   The REC lobe binds tightly to the repeat:anti-repeat duplex of the guide RNA, confirming its..., but they improve cutting efficiency and are important for robust activity in cells (Jiang & Doudna, ... loop to produce SuperFi-Cas9, a high-fidelity variant that still retains high cleavage efficiency (Bravo...correct positioning of the non-target DNA strand is important for stabilizing the active conformation of the... and different techniques all bring their own advantages—and blind spots—to these questions. Each new ...

...book left them wanting to learn more. There are individual paragraphs that may have warranted their own chapters...politicization of science (from climate change deniers to anti-vaxxers), and the very real dangers of quackery,...self-promotion, and personality is playing an increasingly important role in science. The science produced by the quieter...etc.) with that produced by the confident and flamboyant Watsons.” In Part Four, “New Science,” Zimmer...posits the way science is being done has changed significantly over the last decade. He describes the detection... chapters, and certainly chapters that could warrant their own books. Multiple books have been written...

...create specific edits in DNA, there has also been significant work on constructing Cas9 transcriptional activators...upregulation of essentially any gene by fusing mutants of Cas9 deficient in DNA cutting activity to a ... working as expected. Finally, for each gene you want to activate, you should also be ready to test three...Thus, in cases where users have a single gene they want to activate, we would recommend using a cDNA overexpression... the promoter of another gene, thereby driving aberrant transcription. That being said, we generally pick..., Picco G, Gonçalves E, Beaver CM, Migliardi G, Santos R, Rao Y, Sassi F, Pinnelli M, Ansari R, Harper...Addgene Blog Learn how to Choose the Best Cas9 Variant for Your Next Experiment Find out More about Genome-wide...

...future.  Many thanks to our guest blogger Samantha Young. Samantha Young is a Medical Writer with a PhD in... This post was contributed by guest blogger Samantha Young. The use of CRISPR/Cas9 for gene editing has...al., 2013, Cho et al., 2014). There are several variants of the CRISPR/Cas9 system floating around, and...and many pre-designed plasmids containing these variants ready for purchase. But what is the easiest and...). In this case you should be able to find your mutant from screening as little as 8-10 pups. The level...PMC3875854. 2. Mashiko, Daisuke, et al. "Generation of mutant mice by pronuclear injection of circular plasmid...

...compelling of these were those from researchers who wanted more time to mine their own data, with some smaller...work this may entail.  So if access to data is important, what data should be included? We should recognize... storing data in a sharable form is indeed a significant effort. Our standards of data recording are highly...encouraging authors to integrate datasets with their resultant figures. Instead of providing a “dead end” figure...touch interfaces where the viewer is more likely to want to manipulate how they view the information. We ...importance of data sharing The reason this topic is important to my lab is three-fold. Firstly, we rely on many...experiments and understand their data. Likewise, we want to contribute to helping others understand ours....

... the brains of living mice both quickly and significantly. Genes under LITE control showed a rise in transcription...epigenetics with the addition of histone modifiers. “We want to allow people to prove the causal role of specific... Möglich and Peter Hegemann described several advantages of the new system: "First, the light-responsive... methyltetrahydrofolate, which are universally abundant. Second, induction of transcription occurs within...in C. elegans and in Plasmodium. For those who do want to apply LITEs for the transformation of neurons...tip: save yourself some time by making an AAV1 supernatant. (The details can be found here.) So tell us,...