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Render Timestamp: 2024-11-01T10:04:53.010Z
Commit: 23cb9f61fe67e1e9093fd644a533c4ff516a6463
XML generation date: 2024-10-19 02:47:20.504
Product last modified at: 2024-06-27T13:37:00.938Z
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PDP - Template Name: Primer Set (ATO)
PDP - Template ID: *******d82e896

SimpleChIP® Mouse Pdk4 Promoter Primers #31195

Filter:
  • ChIP

    Supporting Data

    REACTIVITY M
    Application Key:
    • ChIP-Chromatin Immunoprecipitation 
    Species Cross-Reactivity Key:
    • M-Mouse 

    Product Information

    Product Description

    SimpleChIP® Mouse Pdk4 Promoter Primers contain a mix of forward and reverse PCR primers that are specific to a region of the mouse pyruvate dehydrogenase kinase 4 (Pdk4) promoter. These primers can be used to amplify DNA that has been isolated using chromatin immunoprecipitation (ChIP). Primers have been optimized for use in quantitative real-time PCR using SimpleChIP® Universal qPCR Master Mix #88989. Primers have been tested in conjunction with SimpleChIP® Plus Enzymatic Chromatin IP Kits #9004 and #9005 and ChIP-validated antibodies from Cell Signaling Technology®.

    Product Usage Information

    Directions for Use:

    1. Label the appropriate number of PCR tubes or PCR plates

    compatible with the model of real-time PCR machine to be used.

    PCR reactions should be performed in duplicate and should

    include a tube with no DNA to control for contamination, and

    a serial dilution of a 2% total input chromatin DNA (undiluted,

    1:5, 1:25, 1:125), which is used to create a standard curve and

    determine amplification efficiency.

    2. Add 2 μl of the appropriate ChIP DNA sample to each tube or

    well of the PCR plate.

    3. Prepare a master PCR reaction mix as described below. Add

    enough reagents for two extra reactions to account for loss of

    volume. Add 18 μl of the master PCR reaction mix to each PCR

    reaction tube or well of the PCR plate.

    Reagent Volume for 1 PCR Reaction (20 μl)

    Nuclease-free H2O 6 μl

    5 μM SimpleChIP® Primers 2 μl

    SimpleChIP® Universal qPCR Master Mix #88989 10 μl

    4. Start the following PCR reaction program:

    a. Initial Denaturation: 95°C for 3 min

    b. Denaturation: 95°C for 15 sec

    c. Anneal and Extension: Primer-specific temp. for 60 sec

    d. Repeat steps b and c for a total of 40 cycles.

    5. Analyze quantitative PCR results using software provided with the real-time PCR machine.

    Storage

    Supplied in nuclease-free water at a concentration of 5 µM (each primer is at a final concentration of 5 µM). Store at -20°C.

    Protocol

    Specificity / Sensitivity


    Species Reactivity:

    Mouse

    Background

    The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to either identify multiple proteins associated with a specific region of the genome or to identify the many regions of the genome bound by a particular protein (3-6). ChIP can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors, and DNA repair proteins. When performing the ChIP assay, cells are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. Fragmented chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or quantitative real-time PCR are often used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing (ChIP-Seq), or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8). SimpleChIP® primers have been optimized for amplification of ChIP-isolated DNA using real-time quantitative PCR and provide important positive and negative controls that can be used to confirm a successful ChIP experiment.
    For Research Use Only. Not For Use In Diagnostic Procedures.
    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
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