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Showing: 181 - 200 of 889 results

  1. Addgene: The First Twenty Years

    Type
    Blog Post
    Published
    Dec. 19, 2024, 2:15 p.m.
    ... our ready-to-use recombinant antibodies service, part of the Neuroscience AntiBody Open Resource (NABOR...library of antibody educational resources, including our new Antibodies 101 eBook, the Antibody Guide, and...could use the same principles to provide recombinant antibodies, another plasmid-based product that, with...front of us. So in our last celebratory moment, we wanted to walk through some of our favorite milestones...designing a website users could easily navigate. This meant we could grow accessibility alongside our repository...so easy!” — Nina Jain, Harvard Medical School. We want to give a special thanks to the early-adopter scientists...beginners” on r/labrats, Reddit.  Part of that work meant facilitating the Material Transfer Agreement process...

  2. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    Published
    Aug. 23, 2018, 12:05 p.m.
    ...are numerous GFP variants. Here I use the name ‘GFP’, which refers to mEGFP. This variant was made by introducing...contributed by guest blogger Joachim Goedart, an assistant professor at the Section of Molecular Cytology...jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness (Tsien, 1998). Another...necessary to generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen...that generates the chromophore (Tsien, 1998). Importantly, this reaction requires molecular oxygen and ...Cranfill et al., 2016 and FPbase.org). A number of variants remain fluorescent under acidic conditions, with...N-terminus, but this hardly reduces its size. The substantial size of GFP may interfere with the activity or...

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

    Type
    Blog Post
    Published
    June 25, 2020, 1:15 p.m.
    ... community which is really important to her. Addgenie Shreya Vedantam with our resident dinosaur...school. QC Scientist Alyssa Cecchetelli knew she wanted to get her PhD. “Going back to school was a pretty...in Addgene since he started, said, “the thing I wanted more of [after undergrad] was to learn and speak...may open doors for you, but depending on where you want to go in your career you may not need those doors...her first job at Addgene as an Order Processing Assistant. After a few months, she left school when she ...Technician. “I was so tired of being in school so I just wanted to work,” she said. “School will always be there...mean you’ve failed. “I got a job as a research assistant in a lab at the university that I went to. Making...

  4. Progress Towards a PAM-Free CRISPR

    Type
    Blog Post
    Published
    March 7, 2024, 2:39 p.m.
    ...Notably, the SpRY variant didn’t display higher off-target effects than SpCas9, a pleasant surprise since... PAM requirement, and how these proteins are advantageous in today’s genome editing landscape. The need...anywhere within the RNA will do. Similarly, if you want to knock out a gene, a frameshift mutation introduced...that don’t utilize NGG   There are plenty of Cas9 variants which utilize alternative PAMs. These alternative...looking for more pan-genome PAM flexibility, you’ll want to consider one of several Cas9s that recognize ...compatibility. This alternative PAM requirement adds a significant amount of flexibility compared to a strict single-compatibility...rounds of engineering, they developed the SpRY Cas9 variant (Walton, et. al, 2020). SpRY uses NRN PAMs and ...

  5. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    Published
    Sept. 12, 2017, 1:44 p.m.
    ...recombination software looks for and quantifies the presence of recombinants in recombinase-dependent sequences...experiments to confirm their findings. But no scientist wants to repeat an experiment because of poor reagent ...production process? These questions are especially important when producing multiple vectors in parallel because...serotype of the packaged DNA and look for potential contaminants. The analysis is a 2-step process using Geneious...total reads are most likely to correspond to the contaminant. Each contig is then BLASTed against the NCBI...contaminated. For AAV-68544, the best match for the contaminant is Addgene plasmid #44362. Method validation,... were easily able to identify AAV-44632, the contaminant we mixed into the original AAV sample at 5%. ...

  6. Finding Your Perfect Job After University

    Type
    Blog Post
    Published
    Jan. 12, 2016, 3:30 p.m.
    ... to reassess what I wanted to do and so I applied for a role in Industry. I wanted to get "a real job"...then, and still feel now, that it is important to take advantage of the fact that science is a global ...staff hired in anticipation of the contract suddenly have less work to do than was anticipated and there can... Sutton, located on the edge of London to gain relevant experience. I applied for this summer position...abroad in rural Africa Once this position ended, I wanted to explore another area which I was really passionate...Procedures (SOP’s) and every item I tested was constantly tracked with barcodes and spreadsheets. It was...to be in the lab doing experiments  without the constant fear of failure because I knew the tests worked...

  7. Multifunctional Neural Fibers: Modulation from Gut to Brain

    Type
    Blog Post
    Published
    Aug. 31, 2023, 1 p.m.
    ... tissue temperature proximal to the implanted fibers. As important as its capabilities are the practical...Sands, J., Zhu, W., Tabet, A., Manthey, M., Allen, H., Loke, G., Antonini, M.-J., Rosenfeld, D., Park, ...gut-brain axis (admittedly for self-serving reasons – I want to understand my obsession with the combination ...connection to study on a molecular level. The brain is constantly influenced by signals it receives from the entire...included in them for downstream control – this is important later! Next, additional components are added -... be, as well as other features necessary for implantation in brain tissue. The gut, on the other hand...hand, was the wild west for fiber implants. Due to the soft nature of the intestines and the requirement...

  8. Plasmids 101: Protein tags

    Type
    Blog Post
    Published
    Dec. 11, 2014, 4:26 p.m.
    ...to aid in the expression and purification of recombinant proteins. Tags for stability and solubility ...hurdles to overcome in order to overexpress a recombinant protein? It is not generally in a cell’s best...glutathione S-transferase (GST), which can be fused to recombinant proteins for one-step purification with glutathione...uncharacterized protein, or a protein for which a good antibody has not been developed (and just because your ...your protein has a commercially available antibody, that doesn’t mean it’s a good one), then your first step... an affinity tag to it. The FLAG, hemaglutinin antigen (HA), and c-myc tags have been the workhorses of...depend on your application (see table below). The antibodies available for these tags really are good and ...

  9. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

    Type
    Blog Post
    Published
    Oct. 13, 2022, 1:45 p.m.
    ...repression using Cas9 Don’t want to permanently modify the genome but still want to affect gene expression...post was originally written by Joel McDade and significantly updated in 2022 by Susanna Stroik.  The advent...your experimental target, SpCas9 and its enhanced variants, eSpCas9 and HypaCas9, are great choices. Check...regions of the genome and consider using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) ...added bonus of low off-target effects. The SpG variant, discovered by the Kleinstiver group has a PAM ...Further, this group also characterized a second Cas9 variant, SpRY, which cuts at NRN PAMs with high efficiency...that the desired edit will be performed on all relevant bases within the editing window, for example all...

  10. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    Published
    July 7, 2020, 1:15 p.m.
    ...editing by APOBEC-Cas9 Though the ACE reporter quantifies APOBEC-Cas9 efficiency in real-time, it relies...Harris Lab created a panel of eGFP reporters to quantify the on-target DNA editing efficiency of APOBEC-Cas9...would restore eGFP fluorescence, making it easy to quantify single base editing efficiency by dividing the... et al., 2016). For TREE they engineered a BFP variant that undergoes conversion to GFP after being targeted...deaminase base DNA editor. Specifically the BFP mutant (BFPH66) contains a histidine at amino acid 66....changes the histidine to a tyrosine generating a GFP variant (GFPY66) that has a shifted emission spectra. Thus...the test by infecting HEK-293 cells with the BFP variant, a BE4am-Cas9 base editor, and sgRNA vectors for...

  11. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More

    Type
    Blog Post
    Published
    Aug. 13, 2019, 12:58 p.m.
    ...for every application. Disadvantages include their duplicated (i.e., redundant) genome, their long three-month..., including investigations into environmental pollutants and health, embryo growth, brain function, and...straightforward husbandry and ability to produce abundant, externally fertilized, transparent progeny (Figure...visualize expression data in useful ways. Zebrafish mutants In the 1990’s, two revolutionary large-scale random... mutagenesis screens produced ~1,500 zebrafish mutants that are still used in labs across the globe (Nüsslein-Volhard...which I will come back to) is the one-eyed pinhead mutant, which regulates a developmental pathway implicated... in congenital heart defects and cancer. These mutants are used extensively to study development and disease...

  12. What's Your Organism? Expanding Genomic Tools via the NSF EDGE Program

    Type
    Blog Post
    Published
    May 2, 2019, 12:41 p.m.
    ...Westwood) Dodders are parasitic plants that steal all their food from other plants and are unusual enough to...efficiency to reduce the number of plants you need to grow to get a recombinant because this process is expensive...between gene function and phenotypes in diverse plants, animals, microbes, viruses, and fungi for which...next project. Gymnotiformes and Mormyridae (Jason Gallant) Weakly electric fish are excellent systems to ...are now studied in 40 labs as a great model for plant adaptation and speciation. Their phenotypes are ...great for studying parenting strategies and make important ecological indicators since species live in water...Aiptasia - algae Breviolum minutum symbionts are important models for solutions to the coral reef crisis....

  13. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    Published
    March 21, 2017, 2:30 p.m.
    ...to infect your cells of interest.   Advantages Disadvantages Can carry large transgenes (>8 kb) Potential...is an important feature for your experiment; it must be chosen carefully. Often researchers want a simple...particles into the culture supernatant and you can generally harvest the supernatant containing viruses 24h...cells. These properties make lentiviral vectors fantastic options for delivering shRNA, CRISPR/Cas9 components... cells, it was obvious that its safety was a disadvantage particularly for clinical development. In addition...using them.   Regarding biosafety, it is also important to be aware that lentiviral particles are a powerful...24h after transfection. This supernatant can be stored at 4 °C or can be directly added to your target ...

  14. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    Published
    Jan. 13, 2025, 9:49 p.m.
    ...including indels. Therefore, they engineered a dominant-negative mutant of the protein MLH1, a component of the...produced additive improvements. These variants are worth testing if you want to make your prime editing as efficient...Over 75,000 pathogenic genetic variants have been identified in humans and cataloged in the ClinVar database... potential to correct only a minority of those variants in most cell types. But prime editing, a CRISPR...ones in Addgene’s repository later in the post. Advantages of prime editing Less constrained by PAM sequence...research, the Liu lab created and evaluated 19 PE1 variants with RT mutations known to increase activity, ...came out on top? The Cas9 nickase fused to a pentamutant of M-MLV RT. They called this system PE2, which...

  15. Hot Plasmids April 2018 - Protein Degradation, Nanoscopy, FIRE-Cas9, and Yeast Expression Tools

    Type
    Blog Post
    Published
    April 23, 2018, 2 p.m.
    ...distinguish between protein variants when used with a variant-specific antibody. TRIM21, an E3 ubiquitin ...sequence and RNA expression - but what if you just want to change protein levels? Until recently, no such...customizable protein degradation. Enter TRIM-Away - an antibody-based system developed by the James and Schuh ...recruits ubiquitin and the proteasome to destroy antibody-bound pathogens. Clift et al. found that addition...addition of exogenous TRIM21 protein along with an antibody targeting the protein of interest led to rapid...indicating that TRIM21 overexpression does not aberrantly activate the proteasome. Clift et al. tested ...co-microinjection of TRIM21 protein and the target antibody works well. In larger cell populations, you can...

  16. Proximity Labeling: A Powerful Tool for Protein Complex Purification and Proteomic Mapping

    Type
    Blog Post
    Published
    Dec. 2, 2019, 2 p.m.
    ...directed evolution until they identified a mutant with significantly increased sensitivity that did not affect...compared to the standard E. coli BirA* mutant. RapID consists of this mutant BirA* and an RNA component that ...elucidate these interactions scientists have long used antibody based affinity purification and mass spectrometry...proteins, which can be a challenge for classic antibody based affinity purification/mass spectrometry ...proteins resulting in their biotinylation. The advantage of APEX is its speed, labeling neighboring proteins...calcium uniporter (Martell et al., 2012). It is important to note that the biotin-phenol reagent can be ...original APEX (Lam et al. 2014). They named this mutant APEX2.  Find the APEX2 plasmids! TurboID and minturbo...

  17. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    Published
    Aug. 2, 2016, 2:30 p.m.
    ...plasmid context). The technique is also highly relevant in this age of CRISPR; site-directed mutagenesis...facilitate the repair, the template should be resistant to Cas9 cleavage. If a plasmid contains the template...cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage. Summary of Site Directed...and bacteria are transformed with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are...interest. Gel electrophoresis diagrams of the anticipated restriction analysis results (left) and validation...figure). Template A high purity plasmid prep significantly increases the success rate of site directed ...directed mutagenesis. In addition, you may want to try different concentrations of template (e.g. 0.1-1.0 ng/...

  18. Addgene Launches Partnership with IPI

    Type
    Blog Post
    Published
    June 13, 2023, 1:15 p.m.
    ... ready-to-use recombinant antibodies. The first collection, a set of integrin antibodies, will be available...website and blog, including our antibody guide, antibody protocols, and Antibodies 101 blog series.  Stay tuned...s website this fall. IPI antibodies will come with access to the antibody sequences, in line with the...the rest of Addgene’s recombinant antibody collection, as well as access to IPI educational resources ...resources developed to support use of their antibody tools. Addgene also offers a growing collection of educational...

  19. CRISPR 101: Off-Target Effects

    Type
    Blog Post
    Published
    Feb. 8, 2024, 2:15 p.m.
    ...several options to quantify these events. Whole genome sequencing The only way to quantify off-target effects...there are significant editing events, serving as a proxy for total off-target effects. Quantifying off-targets...CRISPR work, this often means DNA cleavage at unanticipated sites. While off-target effects are nearly impossible...and minimize them, and ways to control for and quantify them when they can’t be avoided. What are off-target... to keep in mind, but likely will not have a significant impact on your data. You are trying to generate...better to reduce off-target effects, but it’s important to not let ‘perfection’ get in the way of ‘good...mispairing of bases. To reduce off-targets, you’ll want to select a Cas enzyme with low tolerance for mismatches...

  20. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    Published
    Nov. 5, 2024, 8:32 p.m.
    ...straightforward — simply spin down the supernatant in a centrifuge to remove any unwanted cellular debris, as you would...produced in multiple different cell types, including plants, mammals, and insects. In this post, we will compare...which we talk about below. Processing The viral supernatant is typically collected over the course of a couple...to 24 hours. Processing the virus-containing supernatant will depend on the virus type. AAV is typically...methods like ultracentrifugation, while lentivirus supernatants are centrifuged and filtered to eliminate any...than a P1 virus. Pro-tip! Centrifuge the viral supernatant in between passages to eliminate dead cells and...of production, saving about a week of work. An important note is that this is only possible because baculoviruses...
Showing: 181 - 200 of 889 results