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We narrowed to 1,342 results for: Nes

Showing: 181 - 200 of 1342 results

  1. Coomassie Purity Stain of Recombinant Antibodies

    Type
    Protocol
    ... to the appropriate well. Pro-Tip Leaving clear lanes between samples will make quantifying the gel easier...Gels . Select Select Next Lane . Repeat for all lanes. Plot the area under the curves for the protein ...Figure 3 Select Analyze . Select Gels . Select Plot Lanes . One graph per lane will appear with peaks representing...

  2. AAV Production in HEK293 Cells

    Type
    Protocol
    ...Reagents Adherent HEK293T cells (ideally AAV-293T clones) Pro-Tip While adherent, these cells are very loosely...Fisher, 12605010) 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment... + 60 mL of 5 M Sodium Chloride + 4 mL of 1 M Magnesium Chloride Close the bottle and mix by inverting...

  3. CRISPR Library Amplification

    Type
    Protocol
    ...short region of DNA: guide RNAs targeting different genes in an organism's genome, for example. Amplification...altering the composition of the library. Bottlenecks, fitness differences, and plasmid recombination can all ...being lost from the pool and/or increasing the skewness of the pool. 8 electroporation cuvettes (BioRad...

  5. General Transfection

    Type
    Protocol
    ...This approach can be adapted for different cell lines and different transfection reagents. Workflow Timeline...cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 6 cells in a T75 flask in a volume...

  6. Immunocytochemistry

    Type
    Protocol
    ... fluorescent secondary antibody. This protocol outlines the steps for fixing and labeling HeLa cells for...and follow your institution's laboratory safety guidelines for disposing of waste in the appropriate container...

  7. Kit Free RNA Extraction

    Type
    Protocol
    ...dissociation of the nucleoprotein complexes. The effectiveness of your RNA isolation will depend on how effective...

  8. Deep Dive: qPCR

    Type
    Blog Post
    ...profiles (Dwight, 2011).  Reference Genes  Choosing a set of reference genes deserves more attention than it...want to ensure that your reference genes are suitable for your cell lines and/or tissue types.  Please do...reference genes, plural. Your data will be much improved if you move to using multiple reference genes whenever... multiplex your reference genes to save on plate space. For many cell lines and tissue types, appropriate...and most machines have a protocol that automatically performs the normalization step.  Machines Most general-use...researcher defines log-linear phase Liu and Saint No Raw Data Reference and target genes can have...previously-generated validation data, be aware that different machines, samples, and treatments may affect the assay....

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

    Type
    Blog Post
    ... epitope tagging of a large cohort of genes (e.g. tagging genes representing a protein super family). ...generation of clonal knock-in lines that we now use for IP and ChIP assays. Tag 96 genes at a time using Custom...knock-in for 96 different genes in a single go (Figure 3). We were able to tag >30 genes/plate in the first ...Derive clonal lines by single-cell deposition in 96-well plates: Characterize clonal lines by ICC and PCR...designed for ~250 genes, we have had about 30% tagging success, although many of these genes were expressed...be used at high throughput because numerous cell lines can be generated, each with a different tagged protein...ChIP assays. Creating epitope-tagged clonal cell lines remains a challenge as it relies on inefficient ...

  10. Cellosaurus: A Cell Line Information Database

    Type
    Blog Post
    ...community – particularly ones that help address ongoing problems, like cells lines. A 2014 study in Science...145,000 cell lines. Cellosaurus Cellosaurus seeks to identify and describe all cell lines used in research...and dog cell lines is included when available, and can be submitted via email for cell lines profiled in...Cellosaurus also identifies problematic cell lines: ones either suspected to be contaminate or easily ...as commonly misidentified lines. Browsing this feature to know if your lines may be especially vulnerable...to help you identify proximal lines you care about. If you have cell lines without a certified origin (...Science found that approximately one-third of cell lines used in scientific research are misidentified! Rigor...

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

    Type
    Blog Post
    ...70% of human genes have a counterpart in zebrafish. Understanding the function of these genes in fish will...Additional mutagenesis projects have since been initiated and today thousands of mutations in genes affecting...zebrafish Random mutagenesis screens provided a major toehold into understanding how genes control biology..., making them ideal for studying vertebrate embryogenesis. To illustrate some of the reasons that zebrafish... is to examine the consequences of manipulating genes. One reason for the success of zebrafish as a model...studies, in which, for example, the activity of all genes in response to an experimental treatment can be ...This can reveal previously unknown functions for genes, and lays the groundwork for understanding the gene...

  12. Fluorescent Proteins 101: Luciferases

    Type
    Blog Post
    ...reaction is not fluorescence! Chemiluminescence, which includes all bioluminescence, is the production of light.... Table 1: Properties of luminescence versus fluorescence   Luminescence Fluorescence Source of ...needed — just the luminescent substrate. This approach preserves the advantages of luminescent assays (low ...For most people, the most familiar source of bioluminescence is the firefly. Along with charming nighttime...releasing light. Created with BioRender.com. Bioluminescent reporters require a chemical substrate (and...a laser in fluorescence assays), which means luminescence assays have lower background levels and improved...luciferases Although firefly luciferase was the first bioluminescent reporter to be cloned and remains popular, ...

  13. Technical Design of a Western Blot

    Type
    Blog Post
    ... common chemiluminescent methods for detection (though the enzymes used for chemiluminescence can be conjugated...classes of reporters: chemiluminescence enzymes and fluorescent proteins. Chemiluminescence enzymes are cheap...Direct westerns are simpler and faster than indirect ones and can be used in highly quantitative assays. On...appropriate for the target(s) and transfer method. Membranes The two membrane options are PVDF and nitrocellulose...the transfer: you’ll need to pre-wet most PVDF membranes before the transfer and allow them to dry afterwards...has higher background noise than nitrocellulose membranes. Pro tip! You can visualize proteins on a PVDF...reprobing, unless you buy specialty nitrocellulose membranes specifically developed to allow stripping. And...

  14. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

    Type
    Blog Post
    ...antibiotic resistance genes. Genes that are targets of antibiotics are often essential genes which creates a.... ICEBs1 carries the genes needed for conjugation, but can also carry cargo genes, like Mobile-CRISPRi...essential genes were less represented while bacteria containing guides targeting non-essential genes remained...parts: a plasmid expressing the Tn7 transposition genes (tnsA, tnsB, tnsC, tnsD, and tnsE), and a plasmid...interest following IPTG induction of conjugation genes and bi-parental mating. Following excision from ...components and a plasmid with the Tn7 transposition genes are transferred to a recipient bacteria strain of...colleagues used Mobile-CRISPRi to knockdown individual genes in several bacteria associated with human disease...

  15. Uncovering Ancestral Bias in CRISPR Research

    Type
    Blog Post
    ...that for approximately 40% of the identified genes, the genes were not expressed at all. This unexpected... and germline sequence variation in cancer cell lines took them down unexpected paths. Though this research...researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic dependencies of individual...if they could identify the ancestry of the cell lines (European, East Asian, Indigenous American, Africa...each ancestry group. Since about 90% of the cell lines used in DepMap CRISPR screens are of either European...for 994 of the Cancer Dependency Map cancer cell lines (left). Heatmap indicating the breakdown of ancestry...groups were differentially dependent on certain genes. This didn’t prove to be correct, either. However...

  16. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    ...antibody’s labeling of the cell lines to the -omics data. The cell lines with higher expression of the ...approaches are commonly used to produce KO cell lines, some of which are commercially available. Various... – you’ll need to ensure that the parental cell lines of your chosen KO line are likely to express the...Correlation with RNA or Proteomic data from multiple cell lines/ types with different expression Flow cytometry ...combination with other approaches. If several cell lines express the target at different levels, it is possible...assessed through its ability to separate the cell lines based on expression of the target. In this case,...should have more antibodies labeling them; the cell lines with lower expression should have less antibodies...

  17. Common Injection Routes in Mice

    Type
    Blog Post
    ... you wait for the anesthetized mice to breathe in the liquid. Restraint: anesthesia Location: nose Gauge...Intradermal Anesthesia Between skin layers 26 < 0.05 mL Advanced Intrathecal Anesthesia Spinal cord ...Intratracheal Anesthesia Trachea 20–23 (tubing) < 0.07 mL Advanced Intranasal Anesthesia Nose n/a < 0.05...be less stressful, because the mice are under anesthesia, but they are not recommended for tumorigenic... required. Restraint: mechanical restraint or anesthesia Location: tail vein or retro-orbitally Gauge:... the muscle. Common uses include administering vaccines, pain meds, or other drugs. Identifying the proper...sciatic nerve. Mice likely experience some muscle soreness after injection, like you might after getting ...

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

    Type
    Blog Post
    ...reusing pipettes for multiple cell lines rather than using disposable ones or reusing gloves. The serum is... technique, PCR primers specific to the 16s RNA genes of various commonly infecting mycoplasmas are used...sensitivity but low specificity (Degeling, 2012). Bioluminescence assay kits are available from different biotech...which trigger cellular mechanisms that generate luminescence. This can then be detected by a luminometer.... for cell culturing and maintenance of the cell lines. Providing such an environment for the lab facility...Wear a clean lab coat and mask while handling cell lines. Avoid discussion with other lab members, sneezing...wearing PPE. Photo by Kate Harten DeMaio. Cell lines and reagents Examine your cell line stock for mycoplasma...

  19. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    ... to insertional mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you want.... Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene...of research purposes, like creating stable cell lines, delivering CRISPR/Cas9 to cells for genome editing...packaging plasmid contains Gag, Pol, Rev, and Tat genes. Specifically for our knowledge here, the Pol gene...transcriptase), and IN (integrase). These viral packaging genes are required for the production of lentiviral particles...portion does not contain any of the viral packaging genes, the natural cycle of the lentiviral vector ends...process and episomal forms of non-integrating transgenes. Created with BioRender.com; adapted from Banasik...

  20. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ...important to know the brightness of individual fluorophores. Table 1 describes the brightness of a few commonly...fluorophore brightness levels, these differences are much more subtle (think of brightness levels on your...there are fixable dye stains that react with free amines on and inside the cells and are thus typically ...en-us/resources/bd-spectrum-viewer.       Brightness Apart from the excitation and emission wavelengths...commonly used fluorophores. A fluorophore's brightness refers to the signal strength of the emitted photons...fluorophores have a stronger signal. Table 1: Degrees of brightness for a few commonly used fluorophores in relation...emea.bd.com/panel-design/en/flow-cytometry-fluochrome-brightness-spillover Excitation Laser Color Very Bright...
Showing: 181 - 200 of 1342 results