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We narrowed to 1,591 results for: nin

Showing: 281 - 300 of 1591 results

  1. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    ...For these reasons, we are in the process of transitioning our titering method from qPCR to ddPCR. Droplet...ddPCR) does not require a standard or a reference, meaning saving on reagents (and time)! AAV titering using...droplet digital PCR Droplet digital PCR involves partitioning a PCR reaction mixture into approximately 20,000...ingredients for amplification of the target DNA. This partitioning reduces the number of PCR inhibitors per reaction...replicates are built into the technology. One well (containing thousands of droplets) can be sufficient to capture...dilutions, they are transferred to a new plate containing the mastermix which includes primers and a ddPCR...small channels to create a water-in-oil emulsion containing approximately 20,000 droplets. Each droplet contains...

  2. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    ...another genetic disease more prevalent or life-threatening (cystic fibrosis, hemophilia, muscular dystrophy... years ago when completing her PhD and medical training in Ophthalmology. Gene therapy was in its infancy...Importantly, it is an immune privileged compartment, meaning that it can tolerate the presence of antigens (... Congenital Amaurosis (LCA). Shortly after the cloning of the RPE65 gene, a naturally occurring animal...technology: engineering of new capsid variants, designing expression cassettes to regulate and optimize ...reports suggest that the first clinical study combining these tools may start as early as next year. Twenty... areas of science. This is hopefully only the beginning of a gene therapy revolution and the development...

  3. 15 Hot Plasmids from 2017

    Type
    Blog Post
    ...that have standard cloning sites or gateway compatible cloning sites for easy cloning of your genes of ...interneurons in mice as well as other organisms, opening the possibility of using these tools in virtually...ability to be coexcited along with EGFP while retaining an emission spectrum that is easily separable ...editing, Rees et al. created HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9. They subsequently...reprogramming  Regulation of telomere length with TZAP Maintaining telomere length is important for cellular function...function and genomic stability, as telomere shortening leads to premature aging and cancer. The Denchi ... and repair templates of choice though modular cloning steps. The Voytas lab plans to update the toolkit...

  4. Technical Design of a Western Blot

    Type
    Blog Post
    ... thought about running one, you’ll know there’s a lot of choices to make when designing the experiment...for SDS-PAGE running conditions and will cover most western blot use cases. If you’re running native (non-reduced...of gels  Gel Type Protein sizes Running buffer Running conditions Pros Cons Tris-glycine...Figure 1: The technical decisions to make when designing a western blot.  Psst! Looking for a western...lyse and denature them. If you are intentionally running non-denatured proteins, in what is known as a native...proteins. For this post, we are assuming that you are running a denaturing blot.  There are a number of lysing...cytoplasm or whole-cell lysate. A harsher, SDS-containing lysis buffer, like RIPA, should be considered...

  5. A Needle in a Base-Stack: Cas9 Structural Biology

    Type
    Blog Post
    ...species, explaining why they recognize different guide RNAs. Deep in the REC lobe, the arginine-rich bridge... we can see why: two arginines make base-specific hydrogen bonds to the guanines, but the N (a thymine...guide RNA is bound primarily by the REC lobe. An arginine-rich bridge helix connects the two lobes and helps...stem loops bind along the outside of the protein, spanning both lobes. Stem loop 1, nestled between the REC... (yellow) make base-specific hydrogen bonds to guanines of the PAM in the non-target DNA strand (dark ..., 2022; Sternberg et al., 2015). The correct positioning of the non-target DNA strand is important for...DNA strand, and Cas9 bends the DNA helix by transitioning between the open and closed conformations. This...

  6. Viral Production at Addgene

    Type
    Blog Post
    ...electrophoresis (PAGE) followed by silver staining or SYPRO Ruby staining and the molecular weight and relative...after negative staining. Empty vector particles can be identified after negative staining and appear darker...of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV subjected to ...Microscopy The ratio of empty to full (i.e., genome-containing) AAV particles within representative vector preparations... Trono lab. Cell culture medium (OptiPro SFM) containing lentivirus is first cleared by low-speed centrifugation...followed by centrifugation. Precipitated pellets containing viral particles are then resuspended in PBS, ...

  7. New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression

    Type
    Blog Post
    ...extract, and 5 g/L NaCl) or on LB-agar plates containing glucose and L-arabinose at 37oC for at least ...induced with arabinose and colonies on arabinose-containing plates will be visibly white, and gas vesicles...expression heterogeneity required single cell screening to isolate a clonal cell line capable of producing... gene silencing required addition of chromatic-opening agents to boost expression to appreciable levels...Anabaena GVs in mammalian cells without the shell stiffening protein GvpC, which allowed the GVs to be flexible...vesicle expression and robustness without chromatin-opening agents. It ended up being very straightforward ...cells for a several generations to dilute the unintegrated plasmids (Fig. 2b). Next, perform a short (12...

  8. Bioinformatics at Addgene

    Type
    Blog Post
    ... process and tested it by running the main Nextflow script file (containing our new process) on our local...From the beginning, one of Addgene’s goals was to ensure the scientific community obtained high-quality...pipeline. Benefits of Nextflow for bioinformatics pipelining In many use-cases, including ours at Addgene,...a container is sequestered from other programs running on a computer, so it safely operates in isolation... to the ease of jumping back and forth between running the pipeline locally and in the cloud. Creating...print statement at the end of the script or by opening the files. By working on one process at a time,...and decreased cost compared to purchasing and maintaining our own infrastructure. Our patchwork collection...

  9. New CRISPR Tools: Cas7-11 and PASTE

    Type
    Blog Post
    ... revolutionary discovery, including CRISPR, is turning potential into impact. In molecular and cellular... contradicting their initial assumption. Eugene Koonin and Kira Makarova, longtime collaborators of Omar... the project. Using computational observations, Koonin and Makarova found that Cas7-11 appeared to have...toxicity compared to other RNA editing methods — streamlining experimental design and its potential for clinical...reported no detectable effect on cell viability – meaning there was no measurable cell toxicity – using Cas7...integration was observed in non-dividing cells, meaning that this system wasn’t dependent on the cell’s...sequence and integrate it into any DNA sequence containing the 38-50 bp sequence recognized by the serine...

  10. Deciphering the Mysteries of Behavior with Viral Vectors

    Type
    Blog Post
    ...recordings and lesioning studies have been essential for this task. However, most lesioning methods destroy...destroy areas larger than intended and may unintentionally remove connecting pathways between other distant...function, with far fewer side effects compared to lesioning methods -  they may be the key to further unraveling... brain areas associated) compared to an animal running to escape a threat. Locomotion can thus be used...transgenic animals and viral vectors, scientists are combining genetic manipulations with the forced swim test... as sleep, food intake, mobility/coordination, learning, and memory. We’ll cover these in a  future blog... Vincent Jean Pascoli, and Christian Lüscher. "Refining deep brain stimulation to emulate optogenetic ...

  11. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ...genomic fragment containing the sgRNA target sequence can be placed in multi-cloning site (MCS) between...floating around, and many pre-designed plasmids containing these variants ready for purchase. But what is...post will help get your in vivo research up and running as soon as possible! Selecting your target CRISPR...has been quite successfully adapted for use in screening experiments as well). Therefore, if you’ve started...to attack this gene. Should you cut it at the beginning of the coding sequence to delete the entire gene...previously validated gRNAs at Addgene. In vitro screening for the best gRNA Figure 1: Validation ...plasmid was co-transfected with pX330 plasmids containing sgRNA sequences into HEK293T cells. The fluorescence...

  12. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    ...including vector-insert joining for recombinant plasmid construction (restriction cloning), adaptor ligation...Learn New Plasmid Cloning Techniques Pushing the Limits of DNA Assembly Plasmid Cloning by PCR Resources...ligases are enzymes that seal breaks in DNA by joining 5 ́-phosphorylated DNA termini to 3 ́-OH DNA termini... is highly active in nick ligation and DNA end-joining, but will also efficiently ligate many undesirable...undesirable structures, including substrates containing gaps or DNA base pair mismatches (8-11). For some applications...applications, there cannot be any end-joining activity, and for others, there is a need for the exclusive...DNA Ligase, which only reacts with substrates containing no gaps, and will not join any fragments end-...

  13. Our Most Popular Blog Posts from 2019

    Type
    Blog Post
    ... and find plasmids containing these fluorescent proteins at Addgene. 7. Designing Your Chalk Talk for... Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable...Proteins by Cary Valley, Shreya Vedantam, Michelle Cronin, and Angela Abitua Thanks to a team effort from...101: Positive and Negative Selection for Plasmid Cloning by Jennifer Tsang There are many strategies for...systems and sacB counter-selection) tools used in cloning. 8. Bright Monomeric Fluorescent Proteins: mNeonGreen...chalk talk for an academic job interview including defining your research vision, organizing your vision into...

  14. Antibodies 101: Choosing the Right Isotype

    Type
    Blog Post
    ...secondary antibody) approach?  Staining for multiple targets When staining multiple targets indirectly, ...for a four-antibody panel, staining in parallel    In short, when staining multiple targets, look for...will recognize and bind to your primary antibody. Manning et al. tested a variety of commercially available... was a detection bias of IgG2a > IgG2b > IgG1 (Manning 2012).      Figure 1: Broadly reactive...depends on the application and target. If you are running a basic western blot and probing a single target...mouse tissues, causes a high degree of background staining. This occurs because the anti-mouse secondary ...the tissue sample. There are species-on-species staining protocols that use antibody fragments to block...

  15. Working 9-5: an Addgene Co-op

    Type
    Blog Post
    ...provided with a detailed training schedule that helped me transition easily. Training and responsibilities...responsibilities Through this training, I was gradually introduced to Addgene as a company and given more details on...on what my position entailed. The official training process was lined out to span the first three months...months of my six month employment. In the beginning, I spent a lot of time shadowing members of my team ... what I was to do for my time at Addgene. This training prepared me for the independent work on the QC...concern to the plasmid function. The steps of the training process gave me enough time to become familiar...

  16. Antibodies 101: Beyond Surface Labeling

    Type
    Blog Post
    ...will introduce you to a few useful labeling and staining techniques beyond standard surface labeling. Pro... Pro tip! Many people use 'labeling' and 'staining' interchangeably to refer to any technique that combines...Addgene uses 'labeling' to refer to antibodies and 'staining' to refer to small molecule dyes. Indirect labeling...labeling must be performed only after viability staining and surface labeling are already done. Intracellular... viability. Unlike intracellular labeling, dye staining can be done on live cells. For cellular proliferation...cell, the ester group is cleaved off by enzymes, turning the resulting molecule fluorescent and membrane-impermeant... for specific downstream applications, while maintaining a clean cell surface, dump gating is a useful...

  17. Transferable Skills Guide: Conflict Resolution

    Type
    Blog Post
    ...alleviate those differences, they had a hard time maintaining their friendships. I put “neutral” in quotations...learn how to sell them. Types of Conflict Before explaining my approach to conflict resolution, it’s important... into a gap between teams that nobody is really owning. Conflict in this latter scenario usually manifests...disagreeable, you recognize their feelings as valid. Listening: In one of my favorite books, Michael Ende’s Momo... nearby village (and to the reader) by simply listening. Chances are, you know someone like this, even...conflicts.  Institutional Knowledge: Of the tools I’m mentioning here, this may be the most controversial, so ...also aggravate long-term personal conflicts.  Transitioning a conflict into a collaboration may not be the...

  18. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    ...commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway, leading to nonspecific nucleotide...Furthermore (3), we have developed a framework that, by combining these two strategies allows you to specifically...find the 0.2-0.5% correctly edited alleles, when screening reasonable amounts of single cell clones. Furthermore...Furthermore, CRISPR/Cas9 editing is often bi-allelic, meaning that even if one allele is accurately edited, the... few hundred. From these two steps, you’ll be screening a total of a few hundred clones - Still better...longer feasible to incorporate mutations without screening thousands of clones to find a positive one. So... single cell. We calculated the likelihood of obtaining homo- and heterozygous mutations by multiplying...

  19. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    ...More complex 24-fragment assembly Single insert cloning efficiencies were measured based on the acquisition...Antibiotic selection allowed for high throughput cloning efficiency measurements. We performed additional...suppression of background by antibiotic selection. Screening of transformants by colony PCR confirmed the insertion...presence of every module, destination construct and functioning Type IIS restriction enzyme and DNA ligase, any...obtained if any single component was omitted. Screening for successful Golden Gate Assembly All Golden...researchers can plate larger volumes of the outgrowth containing the transformants onto the selection plate. Figure... Ligase, the mainstay of most biotechnological cloning efforts for over 50 years, prefers ligation of ...

  20. Transferable Skills Guide: External Collaborations

    Type
    Blog Post
    ...that many researchers feel that they lack formal training in a variety of transferable skills. At Addgene...huge asset! As is true for any skill, there is a learning curve for collaborating and it is always a good...perfect platform for practicing initiative and for training in teamwork and collaboration management. For ... email and social media (see the section on “Maintaining Relationships” below). Expand: Collaboration ...complex projects. It is also fun! I really enjoy learning from others and meeting people with different ...was very difficult and somewhat awkward in the beginning. But, as so often happens, one gets better with...people outside the university within the context of learning and, in my experience, mistakes and insecurities...
Showing: 281 - 300 of 1591 results