SimpleChIP ® Human Tyrosinase Promoter Primers

1/2

SimpleChIP ® Human Tyrosinase Promoter Primers

品牌： CST

下载产品说明书

用小程序，查商品更便捷

对比

咨询

参数

反应种属：

Human

产品介绍

产品信息

简单描述

Primer Set for studying Tyrosinase in the research area.

商品描述

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 μl5 μM SimpleChIP® Primers 2 μl2X SYBR® Green Reaction Mix 10 μl4. 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.

研究领域

代谢,神经科学,

应用

反应种属

Human

目标/特异性

Specificity/Sensitivity

Species Reactivity:

Human

背景

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. 1.Orlando, V. (2000) Trends Biochem Sci 25, 99-104. 2.Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33. 3.Agalioti, T. et al. (2000) Cell 103, 667-78. 4.Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4. 5.Mikkelsen, T.S. et al. (2007) Nature 448, 553-60. 6.Lee, T.I. et al. (2006) Cell 125, 301-13. 7.Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47. 8.Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.

研究领域

代谢,神经科学,

制备和贮存

保存方式

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.

数据库链接

Entrez-Gene ID

7299

UniProt ID

P14679

参考图片

PCR product melting curves were obtained for real-time PCR reactions performed using SimpleChIP^® Human Tyrosinase Promoter Primers. Data is shown for both duplicate PCR reactions using 20 ng of total DNA. The melt curve consists of 80 melt cycles, starting at 55°C with increments of 0.5°C per cycle. Each peak is formed from the degradation of a single PCR product.在使用SimpleChIP® Human Tyrosinase Promoter Primers的real-time PCR反应中获得了PCR产物的溶解曲线。数据显示出两个使用20 ng总DNA为模板的重复PCR反应。溶解曲线是由80个溶解循环构成，从55°C开始并且每个循环增加0.5°C。每个峰都是单个PCR产物的解链。

SimpleChIP^® Human Tyrosinase Promoter Primers were tested on DNA isolated from cross-linked cells using the SimpleChIP^® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. Real-time PCR was performed in duplicate on a serial dilution of 2% total input DNA (20 ng, 4 ng, 0.8 ng, and 0.16 ng) using a real-time PCR detection system and SYBR^® Green reaction mix. The PCR amplification efficiency (E) and correlation coefficient (R²) were calculated based on the corresponding threshold cycle (C_T) of each dilution sample during 40 cycles of real-time PCR (95°C denaturation for 15 sec, 65°C anneal/extension for 60 sec).用SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003在交联细胞中分离的DNA来测试SimpleChIP® Human Tyrosinase Promoter Primers。通过real-time PCR检测系统和SYBR® Green reaction mix，取2% total input DNA连续稀释(20 ng, 4 ng, 0.8 ng, and 0.16 ng)，每个浓度做一个重复，进行Real-time PCR。PCR扩增效率（E）和相关系数（R2）的计算基于real-time PCR（95°C变性15秒，65°C退火/延伸60秒） 40个循环过程中每个稀释样品的相应的阈值周期（CT）。

声明：本官网所有报价均为常温或者蓝冰运输价格，如有产品需要干冰运输，需另外加收干冰运输费。

货号：

12970S

一键复制

询价

500ul

选择数量

当前规格1件起购

配送至

预计3-4周送达，快递: 免运费，若需干冰额外收费

询价大包装询价