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SimpleChIP^® Human CCND1 Promoter Primers

SimpleChIP^® Human CCND1 Promoter Primers #12531

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Supporting Data

REACTIVITY

Application Key:

ChIP-Chromatin Immunoprecipitation

Species Cross-Reactivity Key:

H-Human

Product Information

Product Description

SimpleChIP^® Human CCND1 Promoter Primers contain a mix of forward and reverse PCR primers that are specific to a region of the human CCND1 promoter. These primers can be used to amplify DNA that has been isolated using chromatin immunoprecipitation (ChIP). Primers have been optimized for use in SYBR^® Green quantitative real-time PCR and have been tested in conjunction with SimpleChIP^® Enzymatic Chromatin IP Kits #9002 and #9003 and ChIP-validated antibodies from Cell Signaling Technology^®. The CCND1 gene encodes for Cyclin D1, a member of the conserved cyclin family that is often overexpressed in breast cancers.

Product Usage Information

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

2X SYBR^® Green Reaction Mix 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:

Human

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.

Database Links

UniProt ID: P24385

Entrez-Gene Id: 595

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

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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SimpleChIP® Human CCND1 Promoter Primers #12531