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We narrowed to 971 results for: lars

Showing: 201 - 220 of 971 results

  1. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    ...the new fragment thereby generating a complete, circular plasmid that can be easily maintained in a variety...companies now sell fast digest enzymes that can digest large amounts of DNA in as little as 10 minutes, but check...will need to use a phosphatase to prevent re-circularization of the backbone plasmid (see below). You should..., the DNA migrates toward the anode (+ charge). Larger fragments of linearized DNA migrate slower than... to anneal to one another and form a complete, circular plasmid. You then add DNA ligase to covalently...in the transformation due to background re-circularization and contamination from uncut plasmid. 4. Transformation...Additionally, if your final product is going to be very large (>10kb) you might want to use electro-competent ...

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

    Type
    Blog Post
    ...the Liu lab discovered that cellular mismatch repair contributed to a large number of unintended prime ...stability Traditional sgRNAs are protected from cellular degradation by Cas9 binding, but pegRNAs have ...pegRNAs, but combining PE7 with epegRNAs resulted in similar or reduced editing efficiencies, likely because...edits to opposite strands of the DNA and skip the cellular mismatch repair step (Anzalone et al., 2022) PASSIGE...combine prime editing with a recombinase to install large sequences (up to 10 kb) into the genome (Pandey ...deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nature Biotechnology...Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22...

  3. CRISPR in the Clinic

    Type
    Blog Post
    ...are not limited to, ones targeting muscular dystrophy, cardiovascular disease, inflammatory diseases, blindness...TTR is primarily made in the liver. Similarly, for cardiovascular disease approaches targeting cholesterol...et al). CRISPR engineering of CAR-T cells is particularly promising because of its potential for allogenic... polymorphisms (SNPs) in the TTR gene and is molecularly characterized by misfolding of the TTR protein...mutations or introducing a desired edit, but this particular class of treatments has quite the opposite aim...therapy currently in clinical trials is somewhat similar (Hussein et al). It employs Cas9 to cut at two ...two sites within the viral genome to excise a large portion of it. This ongoing trial is sponsored by Excision...

  4. Viral Vectors 101: Chemogenetics

    Type
    Blog Post
    ...receptors The receptor you choose is largely dependent on what cellular response you aim to manipulate. It...pathways, giving rise to the same cellular responses. The cellular response set off by a DREADD depends ...normal brain function. Chemogenetics has become a popular tool in the field as it provides a noninvasive ... where specific tissues, cell types, or even subcellular regions of a neuron need to be targeted. Targeting...nonnative ligands. However, they retain their intracellular interactions and still set off the same pathways...been derived from muscarinic receptors. The most popular agonist for muscarinic receptor derived DREADDs...such as the serotonin 3 receptor, will promote depolarization in neurons. Alternatively, combining with the...

  5. Transgenic Organisms, Cas9 Gene Drives, and Appropriate Safeguards

    Type
    Blog Post
    .... An accidental release would be particularly dangerous for molecular biology and genetics because the...such as malaria and dengue for many years. Since more than a thousand children died of malaria today, ...safeguards and confinement strategies are available? Molecular confinement involves building gene drives that... tropical mosquito vectors of diseases such as malaria and dengue are currently being performed in regions...performed with two or more confinement methods. Since molecular, ecological, and reproductive confinement typically...cost of an accidental release. Science relies on popular support, which in turn depends on widespread trust...shared environment, they must never be used without popular support2. An accidental release could delay real-world...

  6. Interview: Nicola Patron on Plant Synthetic Biology, MoClo, and More

    Type
    Blog Post
    ... as to make plants, and agricultural crops in particular, resistant to disease. They also devise biosensors...biosensors designed to elucidate the molecular interactions that go on between plants and their pathogens...biology Addgene: Tell me about the Golden Gate Modular Cloning (MoClo) Kit. What does it include? Patron...to show that they work well in plants and give similar stability to transcripts as the ones that people...we are collecting sequences that will provide a similar resource for those involved in monocot engineering...workhorse. Tips & tricks Addgene: Do you have any particular tips for those looking to get started with MoClo... parts. That was the motivation for releasing a large parts kit. Once you have so many parts available...

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

    Type
    Blog Post
    ...Resources on Addgene.org Basic Molecular Biology Protocols Protocol Videos Molecular Biology Reference Pages  ...T4 DNA ligase) are critical reagents for many molecular biology protocols, including vector-insert joining...sequencing (NGS) library construction, and circularization of dsDNA (6). Less commonly utilized in vitro...are sensitive to distortions in helix shape (22). Large purine:purine mismatches and most smaller pyrimidine...with products identifiable and quantifiable by capillary electrophoresis (CE) (see below). This method ...novel 16S rRNA gene PCR-ligase detection reaction-capillary electrophoresis assay." Journal of clinical microbiology.../LDR/flow-through hybridization assay using a capillary tube, probe DNA-immobilized magnetic beads and...

  8. Mycoplasma Contamination: Where Does It Come From and How to Prevent It

    Type
    Blog Post
    ... etc. Contamination by humans accounts for the largest source among those mentioned above. The contaminants...and then specific enzymes are used which trigger cellular mechanisms that generate luminescence. This can... in the form of small clusters, apart from the cellular DNA. Fluorescent DNA staining is another DNA staining...mycoplasmas, contamination by other bacteria or extracellular fluorescent signals produced by these bacteria...masks. Make sure to change the gloves and masks regularly instead of keeping them in the pockets of your...preventing mycoplasma growth. Also, prolonged use of a particular antibiotic can induce resistance for other bacteria...cross-contamination. CO2 incubators should be cleaned regularly and dampness of any kind should be avoided. Distilled...

  9. Plasmids 101: Using Transposons in the Lab

    Type
    Blog Post
    ...lentivirus). Large cargos, such as the ~11 kB cDNA for dystrophin, the gene mutated in muscular dystrophy... being inserted into new target sites. This is similar to how retroviruses, like HIV, replicate. Class...can be inserted between the transposon’s LTRs, similar to when packaging viral vectors.   Let’s take a...vertebrates and integrates in human cells at rates similar to retroviral vectors. The hyperactive version ...piggyBac has an integration efficiency in human cells similar to retroviral vectors. The hyperactive PB transposase...with a maximum cargo capacity of ~200 kB of DNA. Similar to piggyBac, Tol2 also prefers to integrate at ...transposons Now that you’ve learned about some of the popular transposon systems, let’s take a look at how they...

  10. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ...colonies that are comprised of cells with a large nucleus, large nucleoli, and little cytoplasm. Positive...oncogenes, such as Myc. Viral vectors also have the largest genomic footprint of iPSC generating methods due...infect dividing and nondividing cells and have a larger cloning capacity (8-10 kb) than other retroviral...factors driven by one promoter. While lentivirus has similar rates of reprogramming when compared to MMLV-derived...transfection efficiency is cell-type dependent and larger plasmids have lower rates of transfection. Minicircles...other methods. This could be a reflection of the large size of the plasmid or silencing of the plasmid ...modifications are used to protect the RNAs from cellular defenses against ssRNA: phosphatase treatment,...

  11. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ... Laboratory of Molecular Biology. Plasmid cloning is an essential part of any molecular biology project...the MRC Laboratory of Molecular Biology. He is currently investigating the molecular biology of synaptic...for plasmid cloning Find our molecular biology reference here Find molecular biology protocols here  ...current cloning techniques involve the assembly of a circular plasmid in vitro, before transforming it into ...transformed into E. coli and plasmid assembly (re-circularization) occurs in vivo. We use XL-10 Gold E. coli,...Javier García-Nafría from the MRC Laboratory of Molecular Biology. Jake Watson is a postdoctoral researcher...investigator scientist at the MRC Laboratory of Molecular Biology. He is interested in neuronal signal transduction...

  12. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    ...guest blogger Pooran Dewari, a postdoc in Steve Pollard’s lab at the MRC Centre for Regenerative Medicine...workhorses of the cell – never work alone in the cellular milieu. It is, therefore, critical to understand...biochemical tasks in the cell. One of the most popular approaches to study protein interactions is the...spectrometry to identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin fragments that ...ambitious projects that require epitope tagging of a large cohort of genes (e.g. tagging genes representing...success of the epitope tagging experiment depends largely on how efficiently these components are delivered... TAG-IN bioinformatics tool.A recent article by Clarke et al shows that crRNAs binding to the template...

  13. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    ...fingertips the tools to probe cellular dynamics and function with extraordinary molecular detail.   References...offer scientists a simple yet powerful way to tag cellular proteins and investigate protein localization,...fluorescence when illuminated with blue light (1). Similar to PA-GFP, scientists later developed red fluorescent...photoactivatable fluorescent protein for your experiment Similar to performing experiments with standard FPs, before... chimeric protein will oligomerize, disrupting cellular function and localization, or leading to protein...to optimize your activation protocol for your particular set up. Applications of photoactivatable fluorescent...live imaging of proteins, organelles, and cells. Similar to FPs, PA-FPs allow for non-invasive labelling...

  14. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    ...cells. They bind to proteins on the surface of extracellular pathogens such as parasites or microbes, or ...(Cruse et al., 2004). Because proteins are much larger than this, it may contain many epitopes that antibodies...antibodies are less likely to cross-react with other, similar proteins due to recognizing a single epitope of...monoclonal antibodies here. Recombinant antibodies Similar to monoclonal antibodies, recombinant antibodies...proteins. The proteins are separated based on molecular weight by SDS-PAGE, and are subsequently transferred...internal control for Western blots, shows that a similar concentration of cell lysate was added to each ...   Enzyme-linked immunosorbent assay (ELISA) Similarly, ELISA is used to detect the presence of a single...

  15. Plasmids 101: E. coli Strains for Protein Expression

    Type
    Blog Post
    ... dcm: Similarly, strains with this mutation are unable to methylate cytosine within a particular sequence...blog, we reviewed the salient features of several popular strains of E. coli for DNA propagation. While great...recombinant protein expression and some of the most popular commercial strains designed to avoid them. Why...products may be toxic to the host when expressed, particularly those that are insoluble, act on DNA, or are...is completely deleted (designated lon or Δlon) similary reduce proteolysis of the expressed proteins. ...coli T5 promoter (found on vectors such as pQE or similar), inducible under IPTG (lac repressor on the pREP4...from these strains is not recommended as these auxillary plasmids may be isolated during the prepping process...

  16. The Stingy Scientist: How the Baby Gel Box Was Born

    Type
    Blog Post
    ...about $10 US dollars worth of glass powder pruchased from a pottery supply house.  These large, cheap aliquots...about energy conservation even before it was a popular topic in the media.  At one point I started to ... purifying cut plasmid DNA from agar slices.  Molecular biologists used this procedure daily before the...of techniques like PCR and DNA synthesis. Most molecular biologists bought the pricey stuff in 50 ul aliquots...science project started when I realized I was pouring large agarose gels for DNA visualization or isolation,...amount of agarose, I could pour a very thin gel on a large glass slide using only surface tension.  It's a ...despite the fact that my lab mates thought this was hilarious.  Birth of the baby gel box Finally, taking conservation...

  17. Plasmids 101: Screens vs. Selections

    Type
    Blog Post
    ...As an example, say you are considering either a cellular screen or a selection. A selection is a process...all known genes are assayed for their role in a particular capacity. Drug screens, on the other hand, are...suppressor screens These types of screens are very similar to forward and reverse screens, except that they.... Another example is using a mouse model of a particular disease as the input population. For these experiments... which are worth considering. RNAi screens are similar to CRISPR, but instead of knocking out the genes...thus not assayable with CRISPR. Another method, popularized in yeast, are temperature sensitive screens. ...temperature to exacerbate a mutant phenotype, very similar to enhancer/suppressor screens. Selections Selections...

  18. Kiran Musunuru on the Newest TALEN Genome-Editing System

    Type
    Blog Post
    ... using the kit to target a large number of genes involved in cardiovascular and metabolic diseases. We...Regenerative Biology is to understand the basis for cardiovascular and metabolic human diseases. They do that ... Addgene: How do you use this kit to target a particular DNA sequence? Musunuru: The kit includes 832 ...been implicated in disease or, in cases where particular variants or mutations have been reported in people...of the genome editing process. The first really popular tool was the zinc finger nucleases. Unfortunately... much more reliable. If you design them for a particular sequence, there is a much better chance they ...power here is that you could potentially sequence a large number of individuals and find several mutations...

  19. Scientific Reproducibility - Focusing on Solutions at the Minisymposium on Reproducibility

    Type
    Blog Post
    ...results. As biological researchers deal with larger and larger datasets and use computer based analysis techniques...the description to watch any sections you’re particularly interested in, but I’d recommend watching the...Reproducibility Project: Cancer Biology have produced similarly low rates of reproducibility. Failed attempts ...money with estimates reaching in the billions of dollars (Freedman et al 2015). Finally, as Pamela Hines... characterised antibodies against all human extracellular proteins with community-based validation. They...modified analyses on published data if you need to. Similarly, FigShare also makes it easier to share data through...on many different levels - it’s hard to process large amounts of information and, from a SciComm perspective...

  20. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    ...readout for subcellular fractionations, a procedure that isolates proteins from different cellular compartments...protein levels relative to standards, detect the cellular location of proteins, and can be a readout for...addition to the samples, a protein standard of known molecular weight in a separate lane can help confirm the...migration resistance and improve separation. For large proteins, reduce the percentage of acrylamide. To... commonly used, PVDF tends to be slightly more popular due to its durability and high binding capacity... and typically takes less than an hour. However large proteins typically do not transfer as well using...could contain unintended cross-reactive proteins. Similarly, if you vary the experimental conditions from ...
Showing: 201 - 220 of 971 results