基本描述

别名

GoldStar 探针一步法 RT-qPCR 试剂盒

英文名称

GoldStar Probe One Step RT-qPCR Kit

储存温度

避光,-20°C储存,避免反复冻融

运输条件

超低温冰袋运输

产品介绍

产品内容：

组分	G665836 100 rxns	G665836 100 rxns	G665836 100 rxns
2×GoldStar Probe One Step Buffer	1.4 ml	1.4 ml	1.4 ml
GoldStar Probe One Step EnzymeMix	100 μl	100 μl	100 μl
50×Low ROX	-	50 μl	-
50×High ROX	-	-	50 μl
RNase-Free Water	1.5 ml	1.5 ml	1.5 ml

产品简介：
本产品是采用探针法（TaqMan，Molecular Beacon等）进行一步法Real-Time RTqPCR的专用试剂盒。使用本产品进行Real Time RT-qPCR反应时，逆转录和定量PCR 在同一反应体系中进行，反应过程中无需添加试剂，无需打开管盖，避免了污染的同时提高了实验效率。本产品的检测灵敏度高，荧光信号强，信噪比高，非常适合于 RNA病毒等微量RNA的检测。其所包含的特殊缓冲系统能使逆转录酶与DNA聚合酶同时发挥最大功效，提高反应效率。使用本产品可以得到更宽广的线性范围，对目的基因定量更准确，重复性好，可信度高。ROX染料用于校正定量PCR仪孔与孔之间产生的荧光信号误差，一般用于ABI、 Stratagene等公司的Real Time PCR 扩增仪。不同仪器的激发光学系统有所不同，因此 ROX染料的浓度必须与相应的荧光定量PCR仪相匹配。

注意事项：

1．本试剂盒中试剂使用前请上下颠倒轻轻混匀，尽量避免起泡，并经短暂离心后使用。

2 ．本产品以RNA为模板进行一步法RT-PCR实验，在操作过程中应避免RNase污染，

建议在专门的区域进行RNA操作，使用专门的仪器和耗材，操作人员带口罩和一次性手套并经常更换手套，实验相关耗材应用0.1％DEPC（焦碳酸二乙酯）水溶液在 37 ℃处理12小时,并高压灭菌30分钟后使用。

3．本试剂盒中的各试剂应尽量避免反复冻融，反复冻融可能使产品性能下降。

4．本试剂盒必须使用特异性引物，引物的选择可根据具体实验来选择，引物设计的好坏直接影响到RT-qPCR反应的结果，设计引物时需考虑GC含量，引物长度，引物位置， PCR产物的二级结构等因素，建议采用专业的引物设计软件进行设计。

5．本试剂盒推荐使用特异性探针，建议采用专业的设计软件进行设计。

使用方法：

以下举例为常规的反应体系和反应条件，实际操作中应根据模板、引物结构和目的片段大小的不同进行相应的改进和优化。（反应液的配置请在冰上进行）

1 ．将RNA模板、引物、 2×GoldStar Probe One Step Buffer 、GoldStar Probe One Step EnzymeMix和RNase-Free Water溶解并置于冰上备用。

2 ．PCR反应体系：

试剂	25 μl反应体系	终浓度
2×GoldStar Probe One Step Buffer	12.5 μl	1×
Forward Primer，10 µM	0.5 μl	0.2 μM 1）
Reverse Primer，10 µM	0.5 μl	0.2 μM 1）
Probe ，10 µM	0.5 μl	0.2 μM 2）
GoldStar Probe One Step EnzymeMix	1.0 μl	/
RNA Template	X μl	10 pg – 100 ng3）
50×Low ROX or High ROX （optional）4）	0.5 μl	1×
RNase-Free Water	up to 25 μl	/

注意：

1）通常引物浓度以0.2 μM可以得到较好结果，可以在0.1-1.0 μM作为设定范围的参考。

2）使用的探针浓度，与使用的荧光定量PCR仪、探针种类、荧光标记物质种类有关，实际使用时请参照仪器说明书，或各荧光探针的具体使用要求进行浓度的调节。

3）通常RNA模板的量以10 pg – 100 ng为参照，因不同物种的模板中含有的目的基因拷贝数不同，可对模板进行梯度稀释，以确定最佳的模板使用量。

4）不同仪器的激发光学系统有所不同，根据使用荧光定量的仪器选择加入50×Low ROX or 50× High ROX。

3.混匀，短暂离心，将溶液收集到管底。

4.RT-PCR反应条件：

步骤	温度	时间	/
逆转录	45℃	10 min	/
PCR预变性	95℃	10 min	/
变性	95℃	15s	30-40个循环
退火/延伸	60℃	45s	30-40个循环

注意：

1）本产品所采用的热启动酶须在预变性95℃、 5-10 min条件下实现酶的活化。

2）建议采用两步法PCR反应程序，若因使用Tm值较低的引物等原因，得不到良好的实验结果时，可尝试进行三步法PCR扩增，退火温度请以 56℃-64℃的范围作为设定参考。

Product content:

Component	G665836 100 rxns	G665836 100 rxns	G665836 100 rxns
2×GoldStar Probe One Step Buffer	1.4 ml	1.4 ml	1.4 ml
GoldStar Probe One Step EnzymeMix	100 μl	100 μl	100 μl
50×Low ROX	-	50 μl	-
50×High ROX	-	-	50 μl
RNase-Free Water	1.5 ml	1.5 ml	1.5 ml

Product Introduction：

This product is a specialized reagent kit for one-step Real Time RTqPCR using probe methods (TaqMan, Molecular Beacon, etc.). When using this product for Real Time RT qPCR reaction, reverse transcription and quantitative PCR are required
Conducted in the same reaction system, there is no need to add reagents or open the tube cap during the reaction process, avoiding contamination
This has improved the efficiency of the experiment. This product has high detection sensitivity, strong fluorescence signal, and high signal-to-noise ratio, making it very suitable for
Detection of RNA viruses and other trace amounts of RNA. The special buffering system it contains can enable reverse transcriptase to interact with DNA polymerase
Maximize the effectiveness and improve reaction efficiency. By using this product, a wider linear range can be obtained, which is beneficial for the target base Due to more accurate quantification, good repeatability, and high reliability.
ROX dye is used to correct the fluorescence signal error generated between wells in quantitative PCR instruments, and is generally used for ABI
Real Time PCR amplification equipment from companies such as Stratagene. The excitation optical systems of different instruments vary, therefore
The concentration of ROX dye must be matched with the corresponding fluorescence quantitative PCR instrument.
matters needing attention：
1. Before using the reagents in this reagent kit, please gently mix them upside down to avoid foaming as much as possible, and use them after brief centrifugation. 2. This product uses RNA as a template for one-step RT-PCR experiments, and RNase contamination should be avoided during the operation process,
2.It is recommended to perform RNA operations in a dedicated area, using specialized instruments and consumables. Operators should wear masks and disposable gloves and frequently change gloves. Experimental consumables should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours and sterilized under high pressure for 30 minutes before use.
3. Each reagent in this kit should avoid repeated freezing and thawing as much as possible, as repeated freezing and thawing may lead to a decrease in product performance.
4. This reagent kit must use specific primers, and the selection of primers can be based on specific experiments. The quality of primer design directly affects the results of RT qPCR reaction. When designing primers, GC content, primer length, and primer should be considered

Due to factors such as location, secondary structure of PCR products, it is recommended to use professional primer design software for design.
5. It is recommended to use specific probes in this reagent kit and use professional design software for design.

Usage:

The following examples are typical reaction systems and conditions. In practical operation, corresponding improvements and optimizations should be made based on the differences in template, primer structure, and target fragment size. (Please prepare the reaction solution on ice)
1. Dissolve the RNA template, primers, 2xGoldStar Probe One Step Buffer, GoldStar Probe One Step EnzymeMix, and RNase Free Water and place them on ice for later use.
2. PCR reaction system:

reagent	25 μl Reaction system	final concentration
2×GoldStar Probe One Step Buffer	12.5 μl	1×
Forward Primer，10 µM	0.5 μl	0.2 μM 1）
Reverse Primer，10 µM	0.5 μl	0.2 μM 1）
Probe ，10 µM	0.5 μl	0.2 μM 2）
GoldStar Probe One Step EnzymeMix	1.0 μl	/
RNA Template	X μl	10 pg – 100 ng3）
50×Low ROX or High ROX （optional）4）	0.5 μl	1×
RNase-Free Water	up to 25 μl	/

Note:

1) Typically, the primer concentration is 0.2 μ M can achieve good results, ranging from 0.1 to 1.0 μ M serves as a reference for setting the range.

2) The concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance. Please refer to the instrument manual or the specific usage requirements of each fluorescent probe for concentration adjustment during actual use.
3) The amount of RNA templates is usually based on 10 pg-100 ng as a reference. Due to the different copy numbers of target genes contained in templates of different species, gradient dilution can be applied to the templates to determine the optimal template usage.
4) The excitation optical systems of different instruments vary, and depending on the instrument used for fluorescence quantification, 50 x Low ROX or 50 x High ROX can be added.

3. Mix well, centrifuge briefly, and collect the solution to the bottom of the tube.
4. RT-PCR reaction conditions

steps	temperature	time	/
Reverse Transcription	45℃	10 min	/
PCR pre denaturation	95℃	10 min	/
denaturation	95℃	15s	30-40cycle
Annealing/Extension	60℃	45s	30-40cycle

Attention:
1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 5-10 minutes.
2) It is recommended to use a two-step PCR reaction program. If good experimental results cannot be obtained due to the use of primers with lower Tm values, a three-step PCR amplification can be attempted. The annealing temperature should be set within the range of 56 ℃ -64 ℃ as a reference.

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

输入批号以搜索分析图谱:

技术文档和文章

g665836_goldstar probe one step rt-qpcr kit.pdf

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