...forces or (2) the result of competing desires or (3) the presence of obstacles that need to be overcome... your science with others.    My favorite quote about science ever comes from an interview in which someone...storytelling business like to say, science is all about navigating conflict. In literary terms, conflict...find that once I sit down and talk to a scientist about their research, these conflicts come out quite naturally... a simple question, like “Have you ever thought about how X works?”, or even by introducing a person that...devices, we sometimes forget the most important thing about science: It’s cool. Like bowties! Using science ...

...signaling pathway is unknown.      Figure 3: Chimeric OptoXR incorporating rhodopsin undergoes...Ultrafast optogenetic control. Nature Neuroscience, 13(3), 387–392. https://doi.org/10.1038/nn.2495 Marshel...which tools to use, and what do you need to know about them to be successful in your experiments? In this...

... is not prose; it is to be straight to the point. 3. Engage the reader Scientists forget that the reader...successful writer. When I originally talked to my PI about becoming a better writer, I was unsure if I should... a manuscript and critiquing it, were not too laborious, but helped build my understanding of how to generate...and the writing workshop. SWPI's writing workshop About a month later I received another email notifying... manuscript, scientists have a tendency to be elaborate and over explain the material. The point of writing...

...to safely get back to our lab work. Figure 3: When a bison wanders into the campsite, leave your...Addgene blog Learn about other Cas proteins Read all of our CRISPR blog posts Read about GeoCas9, another... able to eliminate the need for axenic (pure) laboratory cultures to study these microbes. From 16S rRNA...by flexibly deploying IgnaviCas9 without concern about its stability under harsh conditions. One example...

...last additional experiment requested by Reviewer #3. But why isn’t the plasmid here yet? A quick look ...materials as quickly as possible. (To learn more about why MTAs matter, check out another blog post: Why...provide specific examples if possible Tell them about the Golden Stopwatch! Any organization that deposits...California Institute of Technology Cold Spring Harbor Laboratory  Dana-Farber Cancer Institute  Fred Hutchinson...

...current with the latest and greatest! Addgene resources 3 Tips to Improve HDR Efficiency for CRISPR Editing ...most popular tweet from the conference is shown above. The blog post I mentioned, John Doench’s How to...forward. To do that, we need to learn much more about cellular DNA repair pathways. An emphasis on cellular...at the Keystone meeting had great things to say about CRISPOR, a tool to help you design, pick, and close...

... Find plasmids from this publication at Addgene. 3. Swarts, Daan C., et al. “Argonaute of the archaeon...impaired NgAgo activity, with three mismatches abolishing activity. One-guide faithful NgAgo guide loading... how it’s working in the comments section! Read About NgAgo User's First Impressions Here References ... Additional Resources on the Addgene Blog Read About Cas9 Homolog Cpf1 Check Out High Fidelty Cas9 Mutants...

...scoop from Addgenie A. Max Juchheim in this post. 3. Plasmids 101: E. coli Strains for Protein Production...eyes open for any new technologies that will bring about the next revolution in biological research. We’re...Plasmids 101: Control Plasmids Need help thinking about the types of controls you’ll need for your next ...Addgene Browse Our CRISPR Resources Learn More about Viral Vectors Browse the Repository  ...

...International Publishing. https://doi.org/10.1007/978-3-319-07758-1_2 More resources on the Addgene blog Viral... using biorender.com   If you’re new to talking about viruses, here’s a quick tip on terminology. “Virus...have a growing number of Viral Vectors 101 posts about each type, so be sure to check our blog, or our ...

...fluorophore's excitation spectra around 640 nm (Figure 3), we see that APC-Cy7 is excited at a lower efficiency...cannot be excited at this wavelength. Figure 3: Excitation and emission spectra for APC and APC-Cy7...spectrum is located in a range of wavelengths of about 380–700 nm. Photons of lower wavelengths are higher... 650 nm, APC-Cy7's global excitation maximum is about 750 nm. If we were to use a laser that excites at...optimal excitation happens at higher wavelengths of about 750 nm. Source: https://www.bdbiosciences.com/en-us... we start building your first panel, we'll talk about one last principle of color emission. As you can...our blog post on color compensation to learn more about them. Alright, let's build your first panel! Designing...

...three systems to analyze single cells from a mixture (3): An optics system A fluidics system An electronics...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a different...biosafety cabinet with cloning rings or following labor-intensive limiting dilution protocols? If this sounds...fluorescent microscopy must be used. To learn more about the most suitable fluorescent microscopy technique...a laser and the instrument collects information about the cell’s size, complexity and fluorescence intensity...captured by detectors and provides useful information about the cell. Light that continues in the same direction... forward scatter (FSC) and provides information about the relative size of the cell; larger cells produce...

...preparation, and lead your own research project.  3. Do you prefer being a generalist? Working on a variety... Resources Resources on the Addgene blog Learn about career planning resources What Do I Do Now? Academic...

...Palindromic Repeats, in Pubmed returned 728 articles (3/12/2014). With so many options for CRISPR plasmid ... gRNA design tools Need more questions answered about CRISPRs? Check out the full list of 16 FAQs answered...

...identifier for individual cells. Each cell is labeled with 3-4 tags to increase the diversity of the CellTag library...Additional resources on the Addgene blog Learn more about induced pluripotent stems cells (iPSCS) and reprogramming...

...Reference Rq(R165L) Human M3 muscarinic Arrestin-2/-3 CNO* Increase Arrestin translocation Arrestin signalling...activity. PSAM-5HT3 LGICs pair a PSAM domain with a 5-HT 3 serotonin receptor sodium- or potassium-selective ...K + ions, and activates neuronal activity. Figure 3: PSAM4-based ligand-gated ion channels, their effect...routes. Image courtesy of Ute Hochgeschwender . Table 3. Descriptions of LMOs and their activity in neurons.... However, CNO is back-metabolized to clozapine and other clozapine metabolites and these can have off-target...concentrations of clozapine N ‐oxide (CNO). CNO is a metabolite of the antipsychotic clozapine and seems to be...experiments in this species may be entirely due to back-metabolism of CNO to clozapine, demonstrating the need for...

...tagged with different fluorescent proteins or dyes. 3. Two-photon microscopy Two-photon excitation microscopy...PMID: 18718768. PubMed Central PMCID: PMC2569002. 3. Egen, Jackson G., et al. "Macrophage and T cell dynamics...fluorescence microscope where the light source is mounted above (epi) the specimen and the excitation light passes...technique that allows imaging of living tissue up to about one millimeter in depth. Two low-energy photons ...response, but the study of these dynamics was still laborious as epifluorescence microscopy wasn’t able to give...skin. Two-photon intravital microscopy. The video above depicts the infiltration of neutrophils expressing...

...(i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy techniques   Fluorescent...methods 2, 920-931 (2005). PubMed PMID: 16299477. 3. Eggeling C, Willig KI, Sahl SJ, Hell SW. Lens-based...call Epi-fluorescence microscopes. (See left image above for an example image). Point Scanning Confocal Microscopy... plane to reach the detector and all light from above or below is excluded. Parallelized Confocal Microscopy...due to limitations in light penetration mentioned above (5). Thick static samples Ex: Fixed tissue sections...sectioning. Point scanning confocal microscopes (see above) often provide the highest quality images for this...

...strand completely; it is essential, however, that the 3’ end of the primer corresponds completely to the template...proceed. Usually a guanine or cytosine is used at the 3’ end, and the 5’ end of the primer usually has stretches...stretches of several nucleotides. Also, both of the 3’ ends of the hybridized primers must point toward ...restriction site at the 5’ end of your primer, note that a 3-6 base pair "clamp" should be added upstream in order...amplification process. When designing, if unsure about what nucleotide to put at a certain position within...capabilities. Taking into consideration the information above, primers should generally have the following properties...

...-mediated marker excision, it takes approximately 3-4 weeks to obtain a homozygous, outcrossed knock-in...produced a higher editing efficiency, while for edits 3’ to the PAM sequences corresponding to the opposite...learn more about genome editing with CRISPRs? Check-out Addgene's CRISPR Resources Learn about gRNA design...interest, I encountered low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering...

...assist with restriction enzyme digestion (usually 3-6bp). Restriction Site: Your chosen restriction site...Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and we will add...our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC-3'. Many restriction enzymes do not cut DNA efficiently...more information). Thus, we recommend that you add 3-6 bases upstream of your restriction site to improve... sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but...including the stop codon (5'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC) and then TAAGCA to ...sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with 18bp of homology to the ORF). We now ...