基本描述

别名

SYBR染料法qPCR mix | UltraSYBR 混合物

英文名称

UltraSYBR Mixture

储存温度

-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

　UltraSYBR Mixture是专用于染料法（SYBR Green I）实时荧光定量PCR的预混体系，浓度为2×，包含 GoldStar Taq DNA Polymerase、PCR Buffer、dNTPs、SYBR Green I 荧光染料和Mg2+，操作简单方便。主要用于基因组DNA靶序列和RNA反转录后 cDNA靶序列的检测。

本品所含的荧光染料SYBR Green I 可以与所有的双链DNA结合，使该产品可用于不同靶序列的检测而不需合成特异性标记探针。其中GoldStar Taq DNA Polymerase是一种经化学修饰的、全新高效热启动酶，在常温下没有聚合酶活性，有效避免在常温条件下由引物和模板非特异性结合或引物二聚体而产生的非特异性扩增，酶的激活须在95℃下孵育10分钟。独特的PCR缓冲体系与热启动酶的组合，有效抑制了非特异性的PCR扩增，显著提高了PCR的扩增效率。该产品适用于无需ROX作为校正染料的荧光定量PCR仪，如：Roche LightCycler 480，Roche LightCyler 96，Bio-rad iCyler iQ， iQ5，CFX96。

U665891	Component	5 mL	40 mL	Storage
U665891A	2×Ultra SYBR Mixture	5×1 mL	40×1 mL	-20°C. Avoid freeze/thaw cycle.
U665891B	ddH₂O	5×1 mL	40×1 mL	-20°C. Avoid freeze/thaw cycle

产品特点

1．本产品中使用了全新高效热启动酶GoldStar Taq DNA Polymerase与独特的PCR缓冲体系，显著提高PCR的扩增效率，具有高灵敏度和特异性强的特点。

2．适用于荧光定量PCR检测，能够准确地对目的基因进行定量和检测。

注意事项

1．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．本产品中含有SYBR Green I荧光染料，保存本产品或配制PCR反应液时应避免强光照射。

3．避免反复冻融本品，反复冻融可能使产品性能下降。

4．本品不能用于探针法荧光定量PCR。

5．配制反应液时，请使用新的或者无污染的枪头和离心管，尽量防止污染。

使用方法

以下举例为常规PCR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不同进行相应的改进和优化。

1．PCR反应体系

试剂	50 μL反应体系	终浓度
2×UltraSYBR Mixture	25 μL	1 ×
Forward Primer，10 µM	1 μL	0.2 μM ¹⁾
Reverse Primer，10 µM	1 μL	0.2 μM ¹⁾
Template DNA	2 μl ²⁾	/
ddH2O	up to 50 μl	/

注意：

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

2）通常DNA模板的量以10-100 ng基因组DNA或1-10 ng cDNA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。

3）推荐反应体系为50 μl，也可以根据实际实验需求按比例扩大或者缩小反应体系。

2．PCR反应程序：

注意！本产品预变性反应必须在95℃ 10分钟下完成！

建议采用下表显示的两步法PCR进行程序设定，本程序是以ABI7500荧光定量PCR 仪为例。若因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，三步法操作步骤详见《反应条件的优化》。

步骤	温度	时间	/
预变性	95℃	10 min ¹⁾	/
变性	95℃	15 s	35-40 个循环
退火/延伸²⁾	60℃	1 min	35-40 个循环
融解曲线分析³⁾	/	/	/
/	95℃	15 s	/
/	60℃	1 min	/
/	95℃	15 s	/
/	60℃	15 s	/

注意：

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

2）退火温度请以60-64℃作为设定范围的参考，发生非特异性反应时，可提高退火温度。

3）本程序是以ABI 7500荧光定量PCR仪为参照设定，融解曲线分析请以所使用的荧光定量PCR 仪推荐的程序进行设定。

反应条件的优化

在荧光定量反应条件优化时，应从引物浓度、退火温度、延伸时间等方面进行考虑，以提高反应特异性和扩增效率。

1．反应特异性和扩增效率高的实验体系应具备以下条件：

1）反应特异性高：阴性对照无引物二聚体等非特异性扩增；不产生目的片段以外扩增。

2）扩增效率高：Ct值低；PCR扩增效率高，接近理论值100%。

2．反应条件优化方法：

1）引物浓度：通常引物浓度以0.2 μM可得到较好结果，可以终浓度0.1-1.0 μM作为设定范围的参考。若提高反应特异性，可降低引物浓度；若提高扩增效率，可增加引物的浓度，由此优化反应体系。

2）退火温度：建议采用两步法PCR，退火温度60℃进行反应。若提高反应特异性，可提高退火温度，以60-64℃作为设定范围的参考。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，三步法的退火温度请以56℃-64℃的范围作为设定参考。

3）延伸时间：建议采用两步法PCR，延伸时间1 min进行反应。若提高扩增效率，可尝试将延伸时间增加，或尝试三步法PCR

注意！本产品预变性反应必须在95℃ 10分钟下完成！

三步法荧光定量PCR（本程序是以ABI7500荧光定量PCR仪为例）：

步骤	温度	时间	/
预变性	95℃	10 min ¹⁾	/
变性	95℃	10 s	35-40 个循环
退火	56-64℃ ²⁾	30 s	35-40 个循环
延伸	72℃	32 s ³ ⁾	35-40 个循环
融解曲线分析⁴⁾	/	/	/
/	95℃	15 s	/
/	60℃	1 min	/
/	95℃	15 s	/
/	60℃	15 s	/

注意：

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

2）无法得到理想的扩增效率时，适当降低退火温度；发生非特异性反应时，提高退火温度。

3）若需提高反应扩增效率，可适当增加延伸时间。

4）本程序是以ABI 7500荧光定量PCR仪为参照设定，融解曲线分析请以所使用的荧光定量PCR 仪推荐的程序进行设定。

UltraSYBR Mixture is a premixed system specifically designed for real-time fluorescence quantitative PCR using the dye method (SYBR Green I), with a concentration of 2 x, containing GoldStar Taq DNA Polymerase, PCR Buffer, dNTPs, SYBR Green I fluorescent dye, and Mg2+. The operation is simple and convenient. Mainly used for detecting genomic DNA target sequences and cDNA target sequences after RNA reverse transcription.
The fluorescent dye SYBR Green I contained in this product can bind to all double stranded DNA, making it suitable for detecting different target sequences without the need for synthesizing specific labeled probes. Among them, GoldStar Taq DNA Polymerase is a chemically modified, novel and highly efficient hot start enzyme that has no polymerase activity at room temperature, effectively avoiding non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature. Enzyme activation requires incubation at 95 ℃ for 10 minutes. The unique combination of PCR buffer system and hot start enzyme effectively inhibits non-specific PCR amplification and significantly improves the amplification efficiency of PCR. This product is suitable for fluorescence quantitative PCR instruments that do not require ROX as a correction dye, such as Roche LightCyster 480, Roche LightCyler 96, Bio rad iCyler iQ, iQ5, CFX96.

U665891	Component	5 mL	40 mL	Storage
U665891A	2×Ultra SYBR Mixture	5×1 mL	40×1 mL	-20°C. Avoid freeze/thaw cycle.
U665891B	ddH₂O	5×1 mL	40×1 mL	-20°C. Avoid freeze/thaw cycle

Product features
1. This product uses a new and efficient hot start enzyme GoldStar Taq DNA Polymerase and a unique PCR buffer system, significantly improving the amplification efficiency of PCR with high sensitivity and strong specificity.
2. Suitable for fluorescence quantitative PCR detection, it can accurately quantify and detect target genes.
Matters needing attention
1. Before use, please gently mix upside down to avoid foaming, and use after briefly centrifugation.
2. This product contains SYBR Green I fluorescent dye. When storing this product or preparing PCR reaction solution, avoid strong light exposure.
3. Avoid repeated freezing and thawing of this product, as repeated freezing and thawing may cause a decrease in product performance.
4. This product cannot be used for probe based fluorescence quantitative PCR.
When preparing the reaction solution, please use new or non polluting gun heads and centrifuge tubes to prevent contamination as much as possible.

Usage
The following are examples of conventional PCR reaction systems and reaction conditions. In practical operation, corresponding improvements and optimizations should be made based on different templates, primer structures, and target fragment sizes.
1. PCR reaction system

Reagent	50 μL Reaction system	Final concentration
2×UltraSYBR Mixture	25 μL	1 ×
Forward Primer，10 µM	1 μL	0.2 μM ¹⁾
Reverse Primer，10 µM	1 μL	0.2 μM ¹⁾
Template DNA	2 μl ²⁾	/
ddH2O	up to 50 μl	/

Attention:
1) Typically, the primer concentration is 0.2 μ M can obtain good results, with a final concentration of 0.1-1.0 μ M serves as a reference for setting the range.
2) The amount of DNA template is usually based on 10-100 ng genomic DNA or 1-10 ng cDNA 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 template to determine the optimal template usage.
3) The recommended reaction system is 50 μ l. The reaction system can also be scaled up or down proportionally according to actual experimental needs.
2. PCR reaction program:
Attention! The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes!
It is recommended to use the two-step PCR method shown in the table below for program setting. This program takes the ABI7500 fluorescence quantitative PCR instrument as an example. If good experimental results cannot be obtained due to primers with low Tm values, a three-step PCR amplification method can be attempted. The operation steps of the three-step method are detailed in "Optimization of Reaction Conditions".

Step	Temperature	Time	/
Pre denaturation	95℃	10 min ¹⁾	/
Denaturation	95℃	15 s	35-40 cycles
Annealing/Extension ² ⁾	60℃	1 min	35-40 cycles
Analysis of Fusion Curve ³ ⁾	/	/	/
/	95℃	15 s	/
/	60℃	1 min	/
/	95℃	15 s	/
/	60℃	15 s	/

Attention:
1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.
2) The annealing temperature should be set at 60-64 ℃ as a reference range. When non-specific reactions occur, the annealing temperature can be increased.
3) This program is based on the ABI 7500 fluorescence quantitative PCR instrument as a reference setting. For melting curve analysis, please use the recommended program for the fluorescence quantitative PCR instrument used.

Optimization of reaction conditions
When optimizing fluorescence quantitative reaction conditions, factors such as primer concentration, annealing temperature, and extension time should be considered to improve reaction specificity and amplification efficiency.
1. An experimental system with high reaction specificity and amplification efficiency should meet the following conditions:
1) High reaction specificity: negative control without non-specific amplification such as primer dimer; No amplification beyond the target fragment is generated.
2) High amplification efficiency: low Ct value; The PCR amplification efficiency is high, approaching the theoretical value of 100%.
2. Optimization method for reaction conditions:
1) Primer concentration: Typically, the primer concentration is 0.2 μ M can obtain good results, with a final concentration of 0.1-1.0 μ M serves as a reference for setting the range. If the reaction specificity is increased, the primer concentration can be reduced; If the amplification efficiency is improved, the concentration of primers can be increased, thereby optimizing the reaction system.
2) Annealing temperature: It is recommended to use two-step PCR with an annealing temperature of 60 ℃ for reaction. If the reaction specificity is improved, the annealing temperature can be increased, with 60-64 ℃ as the reference range for setting. If good experimental results cannot be obtained due to the use of primers with lower Tm values, a three-step PCR amplification method can be attempted. The annealing temperature of the three-step method should be set within the range of 56 ℃ -64 ℃ as a reference.
3) Extension time: It is recommended to use two-step PCR with an extension time of 1 minute for the reaction. If the amplification efficiency is improved, it can be attempted to increase the extension time or try the three-step PCR method

Attention! The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes!

Three step fluorescence quantitative PCR (this program takes the ABI7500 fluorescence quantitative PCR instrument as an example):

Step	Temperature	Time	/
Pre denaturation	95℃	10 min ¹⁾	/
Denaturation	95℃	10 s	35-40 cycles
Annealing	56-64℃ ²⁾	30 s	35-40 cycles
Extension	72℃	32 s ³ ⁾	35-40 cycles
Analysis of Fusion Curve ⁴⁾	/	/	/
/	95℃	15 s	/
/	60℃	1 min	/
/	95℃	15 s	/
/	60℃	15 s	/

Attention:
1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.
2) When the ideal amplification efficiency cannot be achieved, appropriately reduce the annealing temperature; When non-specific reactions occur, increase the annealing temperature.
3) If it is necessary to improve the efficiency of reaction amplification, the extension time can be appropriately increased.
4) This program is based on the ABI 7500 fluorescence quantitative PCR instrument as a reference setting. For melting curve analysis, please use the recommended program for the fluorescence quantitative PCR instrument used.

安全和危险性（GHS）

SDS英文版

技术规格说明书

技术规格说明书

u665891_ultrasybr mixture.pdf

质检证书（CoA,COO,BSE/TSE 和分析图谱)

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

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货号 (SKU)	包装规格	是否现货	价格	数量
U665891-5ml	5ml	期货
U665891-40ml	40ml	期货

UltraSYBR Mixture

库存信息

库存信息

基本描述

安全和危险性（GHS）

技术规格说明书

质检证书（CoA,COO,BSE/TSE 和分析图谱)

溶液计算器

摩尔浓度计算器

稀释计算器

复溶计算器