SimpleChIP ® Human IkappaBalpha Promoter Primers

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SimpleChIP ® Human IkappaBalpha Promoter Primers

品牌： CST

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参数

分子量：

NONE

反应种属：

Human

产品介绍

产品信息

简单描述

Primer Set for studying IkB-a 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

免疫学和肿瘤学,神经科学,

应用

反应种属

Human

目标/特异性

Specificity/Sensitivity

Species Reactivity:

Human

背景

染色质免疫沉淀 (ChIP) 测定法是一种用于在细胞天然染色质背景下探测蛋白质-DNA 相互作用的强大的通用技术 (1,2)。这种测定法用于检测与基因组某个特定区域有关的多种蛋白，或检测某种特殊蛋白结合的基因组的多个区域 (3-6)。ChIP 可用于确定各种蛋白被募集到某个基因启动子上的特定顺序，或用于“测定”整个基因位点上某种特定组蛋白修饰的相对数量 (3,4)。除了组蛋白外，ChIP 测定法还可用于分析转录因子与辅助因子、DNA 复制因子和 DNA 修复蛋白的结合。进行 ChIP 检测时，细胞首先用甲醛固定，甲醛是一种可逆的蛋白-DNA 交联剂，可以“维持”细胞内发生的蛋白-DNA 相互作用 (1,2)。细胞进行裂解，染色质采集后使用超声处理或酶消化法进行碎裂。碎裂的染色质随后使用对某种特殊蛋白或组蛋白修饰具有特异性的抗体进行免疫沉淀。任何与目标蛋白或组蛋白修饰有关的 DNA 序列都会作为交联染色质复合体的一部分进行共沉淀，并且该 DNA 序列的相对数量会在免疫选择过程中达到富集。免疫沉淀后，蛋白质-DNA 交联被逆转，DNA 得以纯化。标准 PCR 或定量实时 PCR 常用于测量通过蛋白特异性免疫沉淀达到富集的特定 DNA 序列数量 (1,2)。或者，ChIP 测定法还可与基因组微芯片（芯片上的 ChIP）技术、高通测序 (ChIP-Seq) 或克隆策略联合使用，它们都能对蛋白-DNA 相互作用和组蛋白修饰进行全基因组分析 (5-8)。SimpleChIP® 引物经优化可用于通过实时定量 PCR 扩增 ChIP 分离的 DNA，并提供用于确认 ChIP 试验是否成功的重要阳性和阴性对照。 Orlando, V. (2000) Trends Biochem Sci 25, 99-104. Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33. Agalioti, T. et al. (2000) Cell 103, 667-78. Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4. Mikkelsen, T.S. et al. (2007) Nature 448, 553-60. Lee, T.I. et al. (2006) Cell 125, 301-13. Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47. Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.

研究领域

免疫学和肿瘤学,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

4792

UniProt ID

P25963

参考图片

PCR product melting curves were obtained for real-time PCR reactions performed using SimpleChIP® IκBα 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 55ºC with increments of 0.5ºC per cycle. Each peak is formed from the degradation of a single PCR product. 从采用SimpleChIP™ Human IkBα Promoter Primers的实时定量PCR中得到PCR溶解曲线。数据是采用20ng总DNA两次PCR得到的。溶解曲线包括80个循环，起始温度位55度，每个循环增加0.5度。单一PCR产物的降解形成一个峰值。

SimpleChIP® Human IκBα 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 (CT) of each dilution sample during 40 cycles of real-time PCR (95ºC denaturation for 15 sec, 65ºC anneal/extension for 60 sec).

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货号：

5552S

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询价

500ul

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