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We narrowed to 836 results for: Eras

Showing: 261 - 280 of 836 results

  1. Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity

    Type
    Blog Post
    Published
    Nov. 5, 2019, 1:28 p.m.
    ...enhanced site accessibility In order to generate a genomic alteration at a specific location, it must be accessible...appropriate for your specific research question. Considerations when choosing your Cas nuclease When choosing... off-target cleavage should be an important consideration during any experiment. Sensitivity Cas9 sensitivity...Comic by Maya Kostman. Another important consideration when choosing a Cas9 is balancing specificity...Jennifer Doudna Labs used targeted mutagenesis to generate hyper accurate Cas9 (HypaCas9). This nuclease ...the Jungjoon Lee Lab used directed evolution to generate Sniper-Cas9 resulting in a Cas9 with improved ...accessibility, specificity, and sensitivity are key considerations in choosing a Cas protein for your experiment...

  2. 15 Years of Addgene: The Top 15 Plasmids

    Type
    Blog Post
    Published
    Jan. 8, 2019, 1:54 p.m.
    ...requested from Addgene psPAX2  - psPAX2 is a 2nd generation lentiviral packaging plasmid from the Didier ...and tet genes. It can be used with 2nd or 3rd generation lentiviral vectors and an envelope expressing...BB)-2A-GFP (PX458). lentiCRISPR v2 - This 3rd generation lentiviral transfer vector also from the Zhang...founding. Find pCMV-VSV-G. pRSV-Rev - This 3rd generation lentiviral packaging plasmid from the Trono lab...lab. Find pRSV-Rev. pMDLg/pRRE - This is a 3rd generation lentiviral packaging plasmid from the Trono lab...This plasmid from David Sabatini’s lab is a 3rd generation lentiviral vector that contains a scrambled shRNA... This vector from the David Root lab is a 3rd generation lentiviral transfer plasmid for cloning and expressing...

  3. Hot Plasmids - March 2020 - base editors, GEVI, MoClo, and optogenetics

    Type
    Blog Post
    Published
    March 3, 2020, 2:15 p.m.
    ...contributed by Jennifer Tsang AAVs are a great gene therapy delivery mechanism, but their small packaging capacity...splitting removes any exogenous sequences and regenerates a native peptide bond at the split site. ...tested the base editors in vivo and achieved therapeutically relevant editing efficiencies in the mouse ...comparable to those currently used in human gene therapy trials. They further tested the base editors by...Niemann-Pick disease type C, directly demonstrating the therapeutic potential for the split base editors. Find the...conversion of CO2 into water and electrical energy generation. Alistair McCormick’s lab recently developed ... Using parts from this kit, the McCormick lab generated knock-out and knock-in mutants, determined protein...

  4. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    Published
    Oct. 9, 2018, 12:21 p.m.
    ...enter postdoctoral training. Similarly, there are several factors to consider when selecting a postdoc mentor...system more, you will have a better idea of how to leverage it to ask new and interesting questions in your...confidence in the reagents and outcomes. This is preferable to finding out you've been doing something all...exploration, discovering new information. There are literally infinite questions one could pursue and experiments...the data and any recent relevant studies in the literature. Plus, presentations benefit from practice. If...better slides that more effectively tell a story? Generally, the answer to the second question is yes. As ...and how your research fits in with the current literature. Finally, if you give your talk or poster to ...

  5. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    Published
    Oct. 27, 2020, 1:15 p.m.
    ...assumption into question. Specifically, CRISPR-generated mutants believed to be knockouts can maintain ...Morgens et al., 2016). Only upon combining the data generated from both methods did a more comprehensive gene...Consequently, I will focus on different approaches and considerations present at the first stage of an RNAi-based... of the RNAi trigger requires a few initial considerations, such as the starting material (DNA-based vector...prevalent method due to ease of use and capacity to generate a long-term RNAi phenotype. Long hairpin RNA The...Importantly, instead of the assortment of siRNAs generated from a long hpRNA trigger, only a single ~21-nt...amiRNA). Although downstream work is required, several vectors can be found at Addgene that could serve...

  6. Antibodies 101: Validation

    Type
    Blog Post
    Published
    March 24, 2022, 1:15 p.m.
    ...validated? This post will go over some of the general considerations you should review before using an antibody...important to know what you are aiming to achieve. In general, these are three core goals of antibody validation...different applications. Here, I want to highlight the general suggestions given by the International Working ...these pillars is that they are intentionally generalizable and not specific to certain applications or ...analysis of transcriptomic or proteomic data from several different tissues or cell types. If your antibody...the brain, but almost none in the liver (A). In general, RNA levels tend to correspond to protein levels...s important to understand if protein:protein interactions could be a factor in your analysis. Example:...

  7. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    Published
    Nov. 5, 2024, 8:32 p.m.
    ...establishment of two cell lines from the insectspodoptera frugiperda (lepidoptera; noctuidae). In Vitro, 13(4), ...packaging plasmids (three packaging plasmids for 3rd generation lentivirus production). The packaging plasmids...Insect cells are a less common option for the generation of viruses, though they are a popular choice ...from the pupa ovarian tissue of fall armyworms (Spodoptera frugiperda). Much like HEK-293T cells, they are...much larger packaging capacity — up to 38 kb! Generally, insect cell production methods produce lower ...versus insect cells for viral production Consideration Mammalian cells Insect cells Viruses AAV and...adeno-associated virus type 2 vectors. Human Gene Therapy, 13(16), 1935–43. https://doi.org/10.1089/10430340260355347...

  8. Plasmids 101: Degron Tags

    Type
    Blog Post
    Published
    March 14, 2024, 1:15 p.m.
    ... molecule). Degron tags can range in size from several kDas up to ~35 kDa and are typically knocked-in...manipulate protein expression. These tags offer several advantages over the alternative systems – CRISPR...interest to the proteasome for degradation. Like several other tags described here, the dTAG uses a small... and widespread usage, a proteolysis-targeting chimera (PROTAC) was developed for HaloTag. PROTACs are...their target protein and an E3 ligase through interacting ligands. After an E3 ligase facilitates ubiquitination... leakiness associated with the system, but the general concept of the system has remained the same. An...predict precisely how a given degron tag will cooperate with your protein of interest and how effective...

  9. Viral Vectors 101: Voltage Indicators

    Type
    Blog Post
    Published
    Jan. 30, 2024, 2:15 p.m.
    ...signal you can generate without disturbing the integrity of the membrane, a concern with several of the previously...we will review how these indicators work, their general applications, and why viruses are essential to ...time from signal input to readable output, which generally exceeds that of the VSD GEVIs. However, since ... to the plasma membrane due to the addition of several targeting domains. The quencher and voltage detector...previously described systems. Several adaptations of this method have been to tag, cage, uncage, and trap...your experimental system.   Figure 2: General overview of chemigenetic tools. A genetically encoded...can be used for chemigenetics, biosensors, and generally as biological tools, our blog has you covered....

  10. Cloning Mammalian Cells with the Agarose Method

    Type
    Blog Post
    Published
    Sept. 7, 2017, 12:17 p.m.
    ...have also been using Addgene CRISPR vectors to generate cell lines with knockouts of specific genes. Many...should be able to keep cells moist and hopefully aerated with a thin layer of molecular biology-grade agarose...sterile truncated pipet tips. Indeed, an experienced operator can pick 48 clones from a 10 cm dish in 15- 20...both Hepes and bicarbonate) is brought to room temperature and placed in the hood. The medium from a 10 ...is capped, swirled and tested for the correct temperature by placing on the wrist or cheek. Within less...the agarose cloning method Test your agarose temperature. Cloning cells using an agarose overlay does ...agarose onto the recipient plate at just the right temperature. If the agarose solution is too hot, the cells...

  11. Great Results Start with Great Standard Curves

    Type
    Blog Post
    Published
    Aug. 15, 2024, 1:15 p.m.
    ...and use a standard curve and provide some general considerations for scientists planning to use them in ...for the known concentrations (standard curve), generating an equation that allows you to determine the ... triplicate. The closeness of the data points generated by replicate values provides useful information.... With the help of graphing software, you then generate a trendline for the data. The appropriate trendline...users aim for an R2 above 0.95.    Additional considerations When running assays that rely on a standard...To calculate the %CV of the standard curve, one averages the individual %CVs for each point on the curve...assay. Try to minimize these.  Review previous literature or results from labmates to get a sense of the...

  12. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    Published
    May 30, 2019, 12:44 p.m.
    ... the Cas9 nuclease in the absence of genomic alterations. These studies utilize a catalytically inactive...dCas9 and targeting gRNA, to identify molecular interactions at this site (Fujita and Fujii, 2013). They ...method can characterize nucleic acid-nucleic acid interactions at a genomic locus (Fujita et al., 2017). CRISPR-mediated...Fujita et al., 2018). Furthermore, the Fujii Lab generated a transgenic mouse line and retroviral expression...when inappropriately expressed can cause facioscapulohumeral muscular dystrophy (FSHD), a disease for ...specific gRNA-dCas9 complex. When combined with next-generation sequencing, this method allows for the recovery...Toshitsugu, et al. "Identification of physical interactions between genomic regions by enCh IP‐Seq." Genes...

  13. INTEGRATE: Bacterial Genome Engineering Using CRISPR-Transposons

    Type
    Blog Post
    Published
    Dec. 7, 2020, 2:53 p.m.
    ...by the Sternberg lab derives from Vibrio cholerae (V. cholerae INTEGRATE - hereafter VchINT) and encodes...and technical considerations Making insertions in bacterial genomes with the V. cholera INTEGRATE system... using Tn5), and as knock-in tools for human therapeutics (e.g. using Sleeping Beauty). Despite their ... CRISPR-Cas, these systems represent the next generation of tools for efficient and programmable insertions...achieved with VchINT, at two different incubation temperatures. Data adapted from Vo et al., 2020. In E...-recombineering, CRISPRa/i, and base editing. Leveraging a unique marriage between distinct biological...

  14. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    Published
    Aug. 30, 2022, 1:15 p.m.
    ...broke down the method into six general steps and outlined the considerations to be made during each step....that occurs depends on the incubation time and temperature, so you may be able to recover good antibody ...fixation*. Organic Solvents Organic solvents, generally acetone, methanol, or ethanol, work by dehydrating...membrane integrity (Fig. 2). For example, saponin interacts with cholesterol in cell membranes and leaves ...Tween-20 are examples of non-ionic detergents that interact with lipids and proteins in membranes and permeabilize...to use your specific antibody for IF, look for general IF protocols for your sample type. Starting with...cross-linking fixation; they require an additional step generally referred to as Antigen Retrieval. I chose not ...

  15. Better Dyeing Through Chemistry & Small Molecule Fluorophores

    Type
    Blog Post
    Published
    Sept. 8, 2016, 2:30 p.m.
    ... and several others can be made fluorogenic using environmentally sensitive fluorophores. Overall, self-labeling...student searching couch cushions for spare change, desperate to extract a few more photons from a sample. Chemical...by Roger Tsien, who showed that the selective interaction between a bisarsenical dye ligand (e.g., FlAsH... lipoic acid ligase, biotin ligase, phosphopantetheinyltransferase) – These enzymes catalyze the covalent...corollary to these innovative labeling strategies, several groups—including mine—have been revisiting the ...PMID: 26000849. 2. Grimm, Jonathan B., et al. "A general method to improve fluorophores for live-cell and...Protein Should I Use? Resources on Addgene.org Generate Fluorescent Protein Fusions Request Plasmids from...

  16. Plasmids 101: Protein tags

    Type
    Blog Post
    Published
    Dec. 11, 2014, 4:26 p.m.
    ...After several decades of trying to address these challenges, researchers have amassed a considerable molecular... overexpress a recombinant protein? It is not generally in a cell’s best interest to overexpress a protein...peptide-based proteosome inhibitors, glutathione S-transferase (GST), which can be fused to recombinant proteins...for affinity and purification An affinity tag, generally a relatively small sequence of amino acids, is...Purification Binding and elution steps use very moderate buffer conditions FLAG DYKDDDD or DYKDDDDK or...interest would eventually get ridiculous (you generally don’t want more tag than protein), 2-3 tags is...IgG-binding domains. TAP has since come to encompass several other tag combinations, though frequently those...

  17. Analyzing CRISPR Editing Results with ICE from Synthego

    Type
    Blog Post
    Published
    May 8, 2018, 1 p.m.
    ...workflows considerably easier, faster, and more efficient than previous methods. Still, generating reliable...sequence you used. The ICE tool will calculate overall editing efficiency and determine the profiles of...detect or analyze complex edits, such as those generated by multiplexed delivery of multiple sgRNAs. Since...particular parameter was automatically adjusted to generate ICE results. If there are no results or there ... the ICE linear regression is computed during generation of the ICE Score, the Pearson correlation coefficient...around the cut site. It shows, base-by-base, the average amount of signal that disagrees with the reference...sequencing bases downstream of the cut. Summary ICE generates NGS-quality CRISPR editing analysis from Sanger...

  18. Save Time with Transient Plant Leaf Transformations

    Type
    Blog Post
    Published
    July 25, 2019, 12:03 p.m.
    ...lines in tissue cultures takes half a year, and generating a transgenic plant can take even longer, a transient...are investigating how the production of cancer therapeutic drugs, vinblastine and vincristine, are regulated...transgenic hairy roots takes at least six months. Generating a transgenic plant takes even longer, is more... that neither a stable cell line or plant is regenerated from the transformed tissue. Gene function is...by working with young seedlings and optimizing several other aspects of the transient transformation procedure...Once we were on the right track, we switched to luciferases to more easily quantify the effect of the different...GUS::tMAS to confirm transformation success. Luciferases are great quantitative reporters and use a substrate...

  19. Sonic Hedgehog? Sleeping Beauty? Learn About the Genes Behind Addgene’s Conference Room Names

    Type
    Blog Post
    Published
    April 2, 2019, 1:44 p.m.
    ...composed of actin and myosin which interact with each other to generate forces that cause cells and muscles...signalling pathway, which is a key regulator of all bilateral animal development. SHH plays a key role in proper...into the right and left hemisphere. Addgene has several plasmids that contain SHH, you can find them all...that are necessary for myosin activity and thus interaction with actin in nonmuscle systems in the fly. Spaghetti...other hand, is important in the formation of the visceral mesoderm which differentiates into the muscles...Embryos that lack bagpipe have severely disrupted visceral mesoderm and subsequently the gut does not form...is required for specification of the heart and visceral muscles in Drosophila." Development118.3 (1993...

  20. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    Published
    April 16, 2020, 1 p.m.
    ...detected on commercial lateral flow strips, similar to pregnancy tests. With lateral flow strips, the presence... to cleave ssRNA or mRNA. It also exhibits a “collateral effect” of non-specific ssRNA cleavage. How does...sample is first amplified using RPA (recombinase polymerase amplification) or reverse transcriptase (RT)-... for nucleic acid detection almost anywhere as lateral flow strips are easy to transport and work rapidly...diagnostics (e.g. pathogen or virus detection), therapeutics and sensitive genotyping. The beauty of SHERLOCK...Cas12a however recognizes a different PAM site and generates 5’ and 3’ staggered ends after dsDNA breaks. The... DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published this in Nature...
Showing: 261 - 280 of 836 results