.... It’s a problem that he considers “one of the greatest challenges of neuroscience.”  Hot plasmids: Twitch...inside cells that are on the move, such as T lymphocytes. Griesbeck and his colleagues began by isolating...version of the protein with as few calcium-binding sites as possible for added sensitivity. As reported in...these Twitch sensor variants were optimized by testing various linker configurations in a large-scale ...and high resolution functional tracking of T lymphocytes, too, making them a versatile tool for application...other people start using it. For us, it’s like a test.” References Thestrup et al. Optimized ratiometric... functional in vivo imaging of neurons and T lymphocytes. Nat Methods. 2014 Jan 5. doi: 10.1038/nmeth.2773...

...research tools. BenchSci collates information about antibodies while FPbase curates information about fluorescent...sometimes formulating new biological concepts. However, rates of reproducibility for preclinical studies, as reported...Project: Cancer Biology have produced similarly low rates of reproducibility. Failed attempts to apply irreproducible... to future work cost both time and money with estimates reaching in the billions of dollars (Freedman ...making it easier to retain searchable, thorough notes on how lab procedures were actually performed, potentially... The publication record will then show that the tested hypothesis is correct (a great success story) even...category include performing experiments with too few replicates leading to suspect statistical significance (...

...reinhardtii. When exposed to blue light, this channel activates neuronal activity by opening and allowing positively...conformational change in the extracellular domain which activates the native signaling cascade of the intracellular...conformational change when exposed to blue-light and activates downstream secondary signaling pathways. Photoswitchable...light, the ligand engages the binding pocket and activates the receptor. Created with BioRender.com. ...factors will help you determine which one(s) are candidates for your experiments.  Wavelengths Most vectors...et al., 2019) for each tool, and you may have to test and optimize the intensities of light sources used... frequencies (Zhang & Cui, 2015). You’ll need to test for these differences when designing your experiment...

...nuclear processes. The latest of these visualization tools is CRISPR-Sirius, the brightest CRISPR-based tool...CRISPRainbow allows for simultaneous visualization of sites on different chromosomes in living cells, the system...PP7, or boxB sequences inserted into the sgRNA generates blue, green, or red colors for labeling. A combination...in CRISPRainbow, they knew they could be good candidates for further development. They inserted an octet...three loci and measure inter-locus distances. They tested this system out on chromosome 19 by targeting 46... the National Academy of Sciences of the United States of America 112 10 (2015): 3002-7. PubMed PMID: ...

...collection of pathogenic Klebsiella and Pseudomonas isolates causing urinary tract infections. This project... for plant compounds to treat complications of diabetes Gloria Otito Izu, PhD Candidate, University of... the major pathophysiological complications of diabetes mellitus. Recombinant human proteins that play...play a role in insulin regulation and diabetes will be expressed from plasmids (find more information on...of the plant compounds on these proteins will be tested. Investigating changes in neuronal activity after...Armenia Gohar Tsakanova is a Senior Researcher who investigates the molecular and cellular etiopathomechanisms...projects progress. Subscribe to the Addgene blog for updates and future collaborations. Many thanks to all of...

...Light-induced cell ablation with MG-2I-dL5** He et al tested out light-induced cell ablation with MG-2I-dL5*...dye and exposed to light stained dead within 30 minutes of exposure. 90 seconds of illumination were sufficient... fish with the dye. Embryos exposed to just 12 minutes of illumination had fewer mCer3-labeled cells in...cells.  He et al also show that MG-2I-dL5** only generates small amounts of non-specific ROS when exposed... red fluorescent photosensitizer protein that generates ROS when illuminated with green light. They found...that KillerRed required longer exposure times (5 minutes vs 10 seconds) to achieve the same level of killing...cell nuclei. The two photosensitizers had similar rates of off-target damage to neighboring cells. TL;DR...

... an entire experiment and delegate tasks to lab mates or to discuss a potential new research direction... regular check-ins with their employees. 15-30 minutes weekly or monthly will usually suffice to keep ...Managers will get a sense of the employee’s workload, updates on current projects, and offer feedback. The employee...anything that is missing.  During the meeting, take notes on the agenda points. At Addgene, we like to do ...Before leaving the meeting, skim back through your notes and pick out the action items. Move them into a ... positive peer pressure to make sure everyone completes their assigned items to keep the project moving...scope” above) or if you can cut it down to thirty minutes. Keep it moving: If you tend to get stuck diving...

...a versatile tool developed for use in prokaryotes and eukaryotes, works via direct activation. In this...conformational change that removes it from lacO sites within the promoter and ceases repression of the... absent, regulatory protein AraC binds O and I1 sites upstream of pBad, blocking transcription. The addition...addition of arabinose causes AraC to bind I1 and I2 sites, allowing transcription to begin. In addition to...CAP) can also stimulate AraC binding to I1 and I2 sites. Supplementing cell growth media with glucose decreases...sensing protein YFI. When light is absent, YFI phosphorylates FixJ, which binds to the FixK2 promoter to ...

...processing, i.e. tRNAs or Csy4 cleavage sites. This eliminates bulk from crRNA expression plasmids resulting...et al   Multiplexed Lentiviral Expression Cassettes lentiviral transduction One vector expresses... own promoter   Kabadi et al Csy4-Cleavable Cassettes transfection gRNAs expressed from a polycistronic...co-transfection of Cas9 Nissim et alTsai et al PTG Cassettes transfection gRNAs expressed from a polycistronic...s ability to process its own pre-crRNA arrays eliminates the need to include multiple promoters to drive...Multiplex gene editing with CRISPR-Cpf1 is one of the latest developments in CRISPR technologies.  It’s a simple...

...What is the universal thing that unites all scientists? Curiosity? Passion? A love of learning? No. Or... Or at least not entirely. What truly unites us all is the frustration of an experiment failing because...experiment (labeling tubes never takes longer than 5 minutes, right?) As a result, I’d always end up stressed...differently.  Tools to Try  Checklists Found in surgical suites, air fields, and military bases, checklists are... at as you work. Draw your plate maps onto your plates, alongside your physical copy. If you’ve got messy...workspace and your workflow can have a huge impact. Test different approaches, like whether it's better for... forever grateful, I started setting aside 3-5 minutes to sit quietly at my bench before the start of ...

...of animal testing and modifications. The premise of the novel involves Dr. Moreau who creates human-animal...acid recognition sites had to be designed each time we wanted to target new genomic sites. The discovery...proteins are characterized by large DNA recognition sites, which makes them highly specific.  Jurassic Park..., CBE and ABE have obviated the indel formation rates that are typically associated with the DSBs and ...that were “affected by CRISPR.”  Prime editing The latest breakthrough in genome editing is prime editing...intracellular pangenesis to the rediscovery of Mendel. Comptes Rendus de l’Académie des Sciences - Series III ...

...DNA-editing software which integrates our database of over 2000 primers. Two 96-well PCR plates are prepared with...samples through our lab using barcoded tubes and plates. The critical pieces of information for a given...functional mutations, antibiotic resistance, restriction sites used during cloning, and most importantly, the predicted... in-frame protein fusions, and multiple cloning sites. In the simplest case we have a standard backbone... purifying the DNA and gives comparable success rates and sequence quality, which is critical for the ...100 µl of glycerol stock to each of 2 96-well microplates (identical copies, see image above). We then ...

...functional units which are transcribed together. Image courtesy of Robert Hurt. Understanding how individual...Figure 2: The three assembly levels of MoClo. Image courtesy of Rob Hurt. What makes MoClo systems so powerful...optimizing a TU is much easier when you can quickly test that part in different contexts and plug different...products. The flexible nature of MoClo allows users to test a variety of different orientations and components...best promoter for a given peptide can easily be tested by generating Level 1 TUs with unique promoters...modifying enzyme) to pair with it can be queried by testing any number in the Level 2 assembly. These pathways...attractive! It allows researchers to assemble and test many variables all at once. CRISPR CRISPR arrays...

...The job of scientists to observe how things work, test theories, and solve problems is supposed to be impartial...ultimately galvanized scientists to rally in Ottawa to protest the “death of evidence,” and to speak out in unprecedented...Nashville, TN, USA). I had never participated in a protest before, so I had no idea what to expect. Admittedly...although there was at least one person vocally protesting us, he was not threatening in any way. Participating...studies beta-cell function and failure in Type 2 Diabetes. Birmingham, AL, USA 33.5207°N 86.8025°W - Jonathan...March, three climate scientists and an economist testified before the House Science Committee about climate...first step, and continue this work by voting for candidates that respect science, learning to communicate...

...several labs were able to decipher the journey of lymphocytes from the bone marrow to the thymus where they... tagged with a fluorescent protein or dye. It separates the cells mechanically in a vibrating nozzle, ...How do T cells and/or Neutrophils travel to the sites of infection or damage (Peters et al. 2008, Chtanova.... 2008, Kamenyeva et al. 2015)? How do blood monocytes patrol blood vessels during immune responses (...Salomonnson et al. 2012). Finally, it is worthwhile to test the photostability of your fluorescent protein when...flank fluorescent proteins in a plasmid with loxP sites such that their expression is turned on or off in... blood vessels and tissues by a population of monocytes with patrolling behavior." Science 317.5838 (2007...

...interesting adaptive immune response that detects and eliminates foreign DNA, which scientists have modified for... used for small precision edits. NHEJ directly ligates the break ends without the need for a homologous...large genomic deletions or inversions with two gRNA sites on the same chromosome. Most multiplex systems enable...most gRNA targeting sequences will have additional sites of partial homology throughout the genome, called...Directed Repair (HDR) Figure 4: Overview of HDR HDR generates specific genomic edits by copying from a DNA donor...mutation, while whole plasmid or linear dsDNA templates work better for large edits or insertions. When..., where Cas9n nicks the non-target strand and generates a 3’ flap. However, the longer pegRNA also encodes...

...liquid media or solid media (on plates). In general, amplification on plates can help to ensure that diversity...cheaper and easier. One library that Addgene distributes with a chemical transformation amplification ...normal transformation protocol, the size of the cuvettes your lab’s electroporator uses, etc. The DNA amount... anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the library, but generally needs...library varies significantly in plasmid size). On plates, each transformed bacteria will be its own colony... different, and you should always experimentally test the protocol you design to ensure that it works ...

...restriction enzymes. These cut outside their restriction sites allowing you to easily create customized overhangs...pooled in one tube and ligated in as little as 30 minutes! Alternatively, you might consider Gibson cloning... vector and your insert, DpnI digest away the templates, and transform the PCR products into E. coli. ...cut and ligation processes happen in a matter of minutes. Cost: Funding is usually limited - what cloning...will require a PCR reaction or two to generate templates for the cloning process. Time spent actually doing..., because they will come with comprehensive and tested protocols, as well as premixed and ready-to-use...construct does not contain the type IIS recognition sites, allowing you to keep the type IIS enzymes in the...

...This post was contributed by guest blogger Tessa Cressey. The highly pathogenic Ebola virus belongs to... into the infected cells. The EBOV polymerase replicates the viral genome and transcribes viral genes.... is encapsidated by the nucleoprotein, NP. NP associates with the viral polymerase, which is formed by...mRNAs are translated) by cellular machinery. NP encapsidates the negative sense, single-stranded RNA minigenome...replicate the minigenome, the viral polymerase initiates RNA synthesis at the 3’ end of the minigenome ...polymerase recognizes the gene start signal and initiates transcription of the reporter gene. The polymerase...that various viral components play in disease. Tessa Cressey is a PhD Student in the labs of Dr. Elke...

...interaction for all of us studying human genes in test tubes and gels). As a freshman in college I studied...beneficial for many systems. How the EDGE Program promotes scientific sharing The EDGE Program is designed...approaches, and infrastructure necessary for direct tests of cause and effect hypotheses between gene function...Caterpillars that eat milkweed generate cardenolides that tastes yucky (and is poisonous) for bird predators - birds...for the study of basic evolution of fungi and eukaryotes and includes the fungus that is killing frogs...No transformation techniques are available yet. Polistes (Amy Toth) These wasps are like the ones that ...actually rare in the animal kingdom. Gasterosteus aculeates (Dan Bolnick) A paper on stickleback fish science...