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We narrowed to 1,635 results for: nin

Showing: 321 - 340 of 1635 results

  1. Making CRISPR Plasmids Using Fragmid

    Type
    Blog Post
    ... them not working, or spend a few weeks designing and cloning the vectors you actually want. But now you...Plasmids 101: Modular Cloning Addgene's CRISPR Guide Plasmids 101: Golden Gate Cloning...designed for a quick, plug-and-play approach to cloning CRISPR vectors (McGee et al., 2024). It reduces...truly is plug-and-play. Because of its modular cloning approach, it’s easy to switch out reporters, Cas... overview and timeline of the Golden Gate (GG) cloning approach. Individual modules and a sample ligation...them into a destination vector via Golden Gate cloning, perform an exonuclease cleanup, transform, plate...experiment. As always, we recommend checking your cloning via a miniprep and restriction digest, and then...

  2. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    ...PCR amplified for cloning into one of three Zeamays codon-optimized Cas9-containing binary vectors. The...introduce you to some of your potential options and cloning methods, but please scroll down for plasmids suitable... can be inserted using Gibson Assembly or SLIC cloning methods. A BsaI-based E. coli multiplexing plasmid...together and drive ordered assembly into a Cas9-containing destination vector. Note: depending on which ...four unique kanamycin-resistant plasmids, each containing a different gRNA target sequence downstream of...digested using BsmBI and ligated into Cas9 or dCas9-containing destination vectors. Destination vector options...), and dCas9-VP64 (transcriptional activator)-containing plasmids. Each destination vector contains GFP...

  3. The time and cost required to make a plasmid

    Type
    Blog Post
    ... a plasmid? Or how much time you have to spend cloning before you can start your experiment? What about...order? Sometimes, it feels like an eternity of cloning, waiting, and repeating before you can finally ...course, these were all 2018 numbers, and the intervening five years, inflation has occurred. Here at Addgene...in science – the time spent by the scientists designing and performing the experiments. This cost varies...and money across the board. Consider alternative cloning strategies There are many strategies beyond the...well) plate! How much do repositories help?   Designing precise calculations for any of the numbers in...Addgene Blog 5 factors to help you choose the right cloning method Find blog posts about scientific sharing...

  4. Transferable Skills Guide: Problem Solving

    Type
    Blog Post
    ...found that many researchers feel they lack formal training in a variety of transferable skills. At Addgene...puzzling through your broader research questions and designing future experiments. Being able to work through...at work.   While I was in graduate school and beginning to research non-academic careers, I always felt...dozens of career advice articles, I settled on defining problem solving skills as: One’s ability to logically...the process, you might need to go back to the beginning, reassess the situation, and try a new strategy...scientific method is a core part of our academic training.  Developing strong problem solving skills is ...challenges we encounter. As you spend time in a lab learning techniques and taking on independent projects,...

  5. Channelrhodopsins with improved light sensitivity for minimally-invasive optogenetics

    Type
    Blog Post
    ...channelrhodopsin-expressing neurons, the channelrhodopsins respond by opening and allowing an influx of ions into the neuron ...s lab and Viviana Gradinaru’s lab used machine learning to design new channelrhodopsins that are more ...channelrhodopsin that’s successful in vivo. Using machine-learning to overcome channelrhodopsin design challenges... Arnold and Gradinaru Labs developed a machine-learning model to help them construct “designer” channelrhodopsins...the library to 1,161 sequences. Thirty of these remaining sequences were selected for characterization with...mice that received ChR2(H134R) did not turn. Left-turning behavior in ChRger2-treated mice ceased when the...Mackey ED, Gradinaru V, Arnold FH (2019) Machine learning-guided channelrhodopsin engineering enables minimally...

  6. It’s Bioelectric! An Exciting Interdisciplinary Field of Research

    Type
    Blog Post
    ...can also affect patterning and in some cases even override genetically encoded patterning. Based upon trends... Bioelectric states can alter cell types and patterning There is mounting evidence that signaling via... even be necessary and sufficient for certain patterning activities of entire tissue types or even organs...Since bioelectrical signals are controlled by the opening and closing of ion channel proteins, they are not...developmental biologists and cancer researchers are beginning to turn their attention to bioelectricity. One...Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis." Development 139.2 (2012):...

  7. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    ...comparison of this one-step staining with nanobodies vs two-step staining with antibodies. Nanobodies...before application to the sample. Staining with this multi-color staining workflow yields similar localization... fusion with a reporter enzyme. For multicolor staining, careful consideration must be used when selecting...different IgG subclasses of 1° for multi-color staining Can be expensive Nanobodies   Recombinant...together. At the bench, this results in a shorter staining protocol because the primary antibody can be “...Nanobodies also simplify multicolor staining experiments because they don’t require using primary antibodies from...below, triple colocalization is possible even when staining cells with three IgG1 antibodies. Antibodies were...

  8. Illuminating Choices: A Guide to Selecting Fluorescent Dyes and Ligands

    Type
    Blog Post
    ...visualization of both organic tissues and cell cultures, opening biological interiors to many inquisitive scientists...select the optimal dye or fluorescent ligand when designing an experiment. You may find yourself defaulting...Fluorescent dyes utilize these fluorophores by combining them with a molecular species with high affinity...affinity for an endogenous molecular target, meaning the dye will be attached to the target through binding...so extra care must be taken to assess if proper staining is achieved. Fluorescent ligands have more flexibility...) can be stably combined with nanoparticles, maintaining intensity at ~85% of its original value after...brightness and high degree of overlap. Dyes for DNA/RNA staining should have high affinity and specificity to nucleic...

  9. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    ...number of challenges ranging from long periods of training to limited job opportunities in academia - the...the main reason most people enter postdoctoral training. Similarly, there are several factors to consider...about how to get the most out of your postdoctoral training experience. If you're going to commit to doing...doing a postdoctoral fellowship, what are you signing up for and how can you do it well? Below, I briefly...and create a timeline for achieving these goals. Planning is often more easily done in reverse. Assuming...before the position would begin- for you, at the beginning of Year 5. Now work backwards. To publish a submitted... critical to get an experimental system up and running very early on. Once your system is working, you...

  10. Viral Vectors 101: Chemogenetics

    Type
    Blog Post
    ...combined with. Combining a PSAM with the IPD of a cation channel, such as the serotonin 3 receptor, will...understand the complex wiring of the brain and the underpinnings of neurological disease, neuroscientists need...a popular tool in the field as it provides a noninvasive method for manipulating cell activity, especially...provided by chemogenetics are instead that it is noninvasive and has longer-lasting effects. Building your...issues can be mitigated by using low doses and running appropriate controls (Jendryka et al., 2019), they...promote depolarization in neurons. Alternatively, combining with the IPD of an anion channel, such as the ...

  11. Meet an Addgenie: Insights into Grad School, Career Paths, and More

    Type
    Blog Post
    ..., I have conducted informational interviews with nine Addgenies. There are a few patterns throughout the... Graduate school: Do I or don’t I? I asked the nine Addgenies about why they did or did not go to grad... had always been a student and I truly enjoyed learning.” Addgene’s Director of Product Management Eric...feeling like I could read a scientific paper from beginning to end and really get it.” On the other hand, ...don’t get your PhD you’re set up for failure. Listening to Kenkel’s story has helped me realize that is... see where my life will take me. I’m also just learning. I have a huge interest in data science,” which...they work at Addgene. I posed this question to all nine of my interviewees. Fabienne Furt, Addgene’s Associate...

  12. Mouse Modeling, Part 1: Genetically Engineered Mice

    Type
    Blog Post
    ...maximum genetic heterozygosity in the population, meaning they more closely mimic the genetic diversity of...be engineered by inserting a targeting vector containing the gene of interest into a zygote, or into embryonic...microinjection of DNA or infection with a viral vector containing the gene construct can be used to insert the ...knockin mice. Co-injection of a targeting vector containing homologous ends to the breakpoint promotes homologous...engineering favors DNA repair via non-homologous end joining, the newly inserted gene may have errors introduced...creating the floxed gene segment. Crossing of mice containing a floxed gene to a Cre-expressing strain (which...post of this series, I’ll discuss crossing and maintaining a mouse strain. Additional resources on the...

  13. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...vector”. Cloning Method Choose from: Gateway cloning, gene synthesis, Gibson cloning, ligation-independent...ligation-independent cloning, restriction enzyme, TOPO cloning, unknown, or other. Bacterial Resistance Enter...possible, we propagate plasmids in the standard cloning strain DH5α. For plasmids with highly repetitive...plasmids, choose the NEB Stable strain. For plasmids containing the ccdB gene, such as Gateway vectors, choose...

  14. Plasmids 101: Gram Negative and Positive Bacteria

    Type
    Blog Post
    ...gram negative bacteria, staining them red or pink. At the end of the staining procedure, gram positive...Streptococcus pneumoniae, while the alcohol used in the staining process bleached another, Klebsiella pneumoniae...expanded on Gram’s discovery, adding a second stain, safranin, as a counterstain to help visualize the bleached...and immediately rinse. Flood the smear with the safranin counterstain for 30 - 60 seconds. Rinse the slide...positive (purple) and Gram negative (red/pink) staining. Image credit: Mark Perkins https://www.flickr.com...check out these resources.   Bruckner, M. Gram Staining. https://serc.carleton.edu/microbelife/research_methods...creating a complex within the peptidoglycan layer, staining the cell purple. When the alcohol is added, it...

  15. Hot Plasmids and Viral Preps - May 2021

    Type
    Blog Post
    ...were used in this study.  The zCas9i cloning kit is a plant cloning kit that contains a series of vectors...Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple antibody-based vaccines...cross-reactivity, antibody candidates underwent affinity screening to select for slow off-rate binders. The best ...protein VP1 is co-expressed with a fusion protein containing VP2C and a protein of interest (POI). The VP2C...with more on the way. S5E2 regulatory element containing vectors developed in the Jordane Dimidschsteind...provide expression selectively in Parvalbumin containing cortical interneurons. We now provide ready-to-use...

  16. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    ... nearly every experiment requiring AAV. Before joining my lab, I had never worked with AAV, so naturally... Two to five days after transfection, the AAV-containing cells and media are harvested and purified. In...duplicated regions like the ITRs. Restriction digest screening for ITR deletion is still necessary. To save time...overlaid. Following ultracentrifugation, the AAV-containing 40% fraction is collected and buffer exchanged...titering by qPCR quantifies the number of genome-containing viral particles that are present in an AAV prep...standard curve, which is generated from an ITR-containing plasmid. This whole process takes ~3 hours to...expression in mammals Use the PEI calculator for planning AAV packaging transfections Find tips for a first...

  17. 10 Basic tips for mammalian cell culture

    Type
    Blog Post
    ...you should always ensure nothing is covering it. Turning on the UV light 3-4 times a week for up to half...microbe-laden aerosols that may enter the hood. Planning your experiments thoroughly before entering the...solves most problems before they occur. With proper planning, you’ll know exactly what materials you’ll need...changes in temperature (especially if you have air conditioning on) so leave them in a sterile incubator. This...make up your master stock, while you can use the remaining vial to grow and freeze additional cells that ... Universität Berlin, Germany. She will soon be joining her PhD lab at the Max Planck Institute of Colloids...the Addgene Blog The Challenges of Cell Culture Cloning Mammalian Cells with the Agarose Method Plasmids...

  18. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    ...are ready to begin the screening process. Want to set yourself up for a screening success? Before you start...’s best to do some planning in the design phase of your experiment for a screening strategy. Restriction...looking for your edit of interest. For all of the screening strategies outlined below, the technique can be... isn’t a multiple of three will do. A popular screening method for knockouts is Tracking of Indels by ...frequency of your sgRNA-Cas9 combo. Pro tip: When designing your PCR, ensure there is at least ~200 base pairs...deletion large enough that it can be visualized by running your PCR on a gel. It’s still paramount that you...using a dual guide system.   Fig. 2: Size screening for large deletion events.   Validating knock-in...

  19. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    ...functional analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key... the plasmid containing the mutation). Similar results can be obtained with PCR screening using the primers...manner. The approach can be used in conventional cloning (to introduce or remove restriction sites), in ...final circular plasmid product pictured here. B) Screening of recovered plasmids for the mutation of interest... vastly facilitates the subsequent process of screening for succesfully mutated clones. Forward and reverse...Taq generate A-overhangs (a feature used in TA-cloning). Such non-complementary bases at the 3’-end interfere...can also form colonies under these conditions. Screening If a restriction site was introduced (or ablated...

  20. Plasmids 101: Choosing an Antibiotic Resistance Gene

    Type
    Blog Post
    ...so that you can specifically isolate bacteria containing your constructs of interest. But does it matter...will lose it during division. These non-plasmid-containing bacteria can compete with and confound the goal...from forming while effectively selecting plasmid-containing bacteria. So… why do some people still use amp...? Zeocin is genotoxic when not 100% inhibited, meaning it will induce mutations in the host/plasmid DNA...Excellent No Bacteria, plants, and eukaryotes Not Tn5-containing cells {% icon icon_set="fontawesome-5.14.0" name...References and Resources References Hwang, J. M., Piccinini, T. E., Lammel, C. J., Hadley, W. K., & Brooks...Antibiotic Resistance Plasmids 101: Blue-White Screening Choosing Your Perfect Plasmid Backbone  ...
Showing: 321 - 340 of 1635 results