PCR DIG Probe Synthesis Kit，PCR地高辛探针合成试剂盒，标记的探针可在Southern杂交后从5-10mg的人基因组DNA检测出单拷贝基因, 是从少量DNA模板制备大量探针的最有效、最经济的方法。现货供应，促销见：http://www.qcbio.com/html/41802.htm

Application

The kit is specially designed for the generation of highly sensitive hybridization probes that are suitable for the detection of low- (single-) copy target sequences. Thus, it should be considered as an alternative to random-primed labeling when either the amount of template DNA is limited or only a specific part of the sequence of the template is required for hybridization. The nucleotide concentration in the PCR DIG Probe Synthesis Kit ensures the identification of single-copy genes in the genomic blots following hybridization to DIG-labeled PCR products. Human single-copy genes are detectable in 10 µg of genomic DNA.
PCR products can be directly generated and labeled from small amounts of genomic DNA (100 ng–1 µg), and subsequently used as hybridization probes.
The PCR DIG Probe Synthesis Kit contains an alkali-labile DIG-11-dUTP formulation. This enables simple removal of the DIG label following chemiluminescent detection, and allows the subsequent rehybridization of blots with multiple DIG-labeled probes.

Background Information

The nonradioactive DIG system uses digoxigenin, a steroid hapten, to label DNA, RNA, or oligonucleotides for hybridization, and subsequent color- or luminescent detection. The digoxigenin is coupled to dUTP via an alkali-labile ester bond. The labeled dUTP can be easily incorporated by enzymatic nucleic-acid synthesis using DNA polymerases.
The polymerase chain reaction (PCR) allows the amplification of minute amounts of DNA to levels above 1 µg. The only prerequisite is that some sequence information of the target sequence is needed in order to synthesize the appropriate primers.
The combination of nonradioactive labeling with PCR is a powerful tool for the analysis of PCR products, and also for the preparation of labeled probes from small amounts of a respective target sequence. The detection sensitivity of these labeled probes can be adjusted by the ratio of the unlabeled nucleotides to DIG-dUTP. Whereas a low ratio of DIG-dUTP to dTTP of 1:19, as in the PCR DIG Labeling Mix, ensures a high-yield amplification reaction with good sensitivity for direct detection, the hybridization to single-copy genes in complex genomes may not be successful. The PCR DIG Probe Synthesis Kit, therefore, contains a high ratio of DIG-dUTP to unlabeled nucleotides of 1:2, ensuring reliable detection of single-copy genes. However, PCR yield may be decreased by up to 50%. Nevertheless, this ratio will yield enough product for several hybridizations.

Contents

1.        Enzyme Mix, Expand High Fidelity, 30 µl (105 U) Enzyme Mix, 3.5 U/µl in storage buffer, 20 mM Tris-HCl, pH 7.5 (25^oC), 100 mM KCl, 1 mM dithiothreitol (DDT), 0.1 mM EDTA, 0.5% Tween 20 (v/v), 0.5% Nonidet P40 (v/v), 50% glycerol (v/v)

2.        PCR DIG Probe Synthesis Mix, 10x conc., 125 µl of a mixture containing dATP, dCTP, dGTP (2 mM each), 1.3 mM dTTP, 0.7 mM DIG-11-dUTP, alkali-labile, pH 7.0

3.        PCR Buffer with MgCl₂, 10x conc., 1 ml Expand High Fidelity Buffer, 10x conc. with MgCl₂

4.        dNTP Stock Solution, 10x conc., 125 µl of a mixture containing dATP, dCTP, dGTP, dTTP (2 mM each), pH 7.0

5.        Control Template, 50 µl (1 ng) plasmid DNA (20 pg/µl) in Tris/EDTA buffer, pH 8.0. The 5-kb plasmid contains the cDNA for the human tissue-type plasminogen activator (tPA)

6.        Control PCR Primer Mix, 25 µl (containing 50 pmol of each primer) control PCR primer 1 and 2 (2 mM each)

Quality

Function test: Each lot of the PCR DIG Probe Synthesis Kit is function-tested in PCR. Amplification products are assayed in genomic Southern blots.
Under PCR conditions described in the pack insert, the control reaction generates an amplification product of 442 bp. Due to multiple incorporations of DIG-dUTP during the PCR process, the molecular weight of the PCR products is significantly increased compared to the unlabeled PCR product.
A specific fragment pattern is detected after hybridization of the PCR product to 10 µg human genomic DNA and chemiluminescent detection as described in the pack insert.

Roche地高辛标记及检测相关试剂盒

名称	编号	产地		规格	价格￥
DIG DNA Labeling and Detection Kit 地高辛DNA标记和检测试剂盒	11093657910	Roche		Kit	8732
	DIG-11-dUTP随机引物法标记DNA, 碱性***酸酶(AP)生色反应检测, 标记探针用于Southern、Northern和斑点杂交以及菌落和噬菌体筛选。组成: 未标记对照DNA1; 未标记对照DNA2; DNA稀释液; DIG-标记对照DNA; 六核苷酸混合物; Klenow 2u/μl; 封闭液; BCIP/NBT; 10×dNTP标记混合物; 抗-地高辛-碱性***酸酶复合物。一个Kit可用于25次标记反应和50次检测(100cm2膜), -20℃保存。
DIG-High Prime DNA Labeling and Detection Starter Kit I 高效DNA地高辛标记和检测试剂盒I	11745832910	Roche		Kit	5457
	比普通DIG随机引物法标记效率高, 操作简单, 可用于Southern、Northern和斑点杂交以及菌落和噬菌体筛选。一个Kit可用于12次标记反应和24次检测反应。组成: 5×DIG-高效标记试剂; 未标记对照DNA, PBR328; DNA稀释液; 抗-DIG-碱性***酸酶复合物; NBT/BCIP储存液; 10×封闭液; DIG定量复试纸条; DIG对照试纸条, -20℃保存。
DIG-High Prime DNA Labeling and Detection Starter Kit II 高效DNA地高辛标记和检测试剂盒II	11585614910	Roche		Kit	5598
	比普通DIG随机引物法标记效率高, 采用CSPD化学发光检测灵敏度更高。一个Kit可用于12次标记反应和24次检测反应。组成: 5×高效DIG标记复合物; 未标记对照DNA, PBR328; DNA稀释液; 抗-DIG-碱性***酸酶连接物; CSPD; 10×封闭液; DIG定量试纸条; DIG对照试纸条, NBT/BCIP储存液; -20℃保存。
DIG DNA Labeling Kit 地高辛DNA标记试剂盒	11175033910	Roche		40T	7753
	随机引物扩增法以DIG-11-dUTP标记DNA; DIG标记探针可用于各种膜杂交。
DIG Nucleic acid Detection Kit 地高辛核酸检测试剂盒	11175041910	Roche		Kit	5165
	NBT/BCIP显色法检测, 灵敏度可达0.1pg同源DNA; 可检测40张100cm2杂交膜。-20℃保存。
DIG Oligonucleotide 3’-End Labeling Kit 寡核苷酸3’末端地高辛标记试剂盒	3353575910	Roche		25T	7432
	通过末端转移酶以DIG-11-ddUTP标记14-100bp的寡核苷酸探针3’端用于各种膜杂交和原位杂交。每个反应可标记100pmol的寡聚核苷酸。
DIG Oligonucleotide Tailing Kit 寡核苷酸加尾地高辛标记试剂盒	3353583910	Roche		25 T	6266
	通过末端转移酶以DIG-11-ddUTP在寡核苷酸探针3’端加一平均长度50bp(10-100bp)的寡核苷酸, 用于DNA/RNA膜杂交和原位杂交。每个反应可标记100pmol的寡聚核苷酸。
DIG RNA Labeling Kit (SP6/T7) 地高辛RNA标记试剂盒(SP6/T7)	11175025910	Roche	2´10T		7592
	由SP6或T7 RNA聚合酶通过转录反应过程标记RNA, 1mg模板可产生约10mg标记RNA用于杂交。-20℃保存。组成: pSPT18 DNA, pSPT19 DNA, 对照DNA1, 对照DNA2, DIG标记对照RNA, NTR标记混合物, 转录缓冲液, RNase抑制剂, 无RNase的DNase I, SP6 RNA聚合酶, T7 RNA聚合酶。