产品简介

 

Hieff® qPCR SYBR Green Master Mix(High Rox Plus)是2×实时定量PCR扩增的预混合溶液。Mix中含有热启动Hieff® DNA Polymerase、SYBR Green I、dNTPs、Mg²⁺及High Rox。使用时，仅需在扩增体系中加入模板和引物即可进行实时荧光定量PCR，大大简化操作过程，降低污染几率。

本品采用的DNA聚合酶配体可以随温度变化实时调节DNA聚合酶活性。配方添加了有效抑制非特异性PCR扩增的因子和提升PCR反应扩增效率的因子，使定量PCR可以在宽广的定量区域内获得良好的线性关系。

 

产品信息

 

货号	11203ES03 / 11203ES08 / 11203ES50 / 11203ES60
规格	1 mL / 5×1 mL / 50×1 mL / 100×1 mL

 

储存条件

 

-25~-15℃保存，有效期18个月。本品避免反复冻融。产品中含有荧光染料SYBR Green I，保存或配制反应体系时需避免强光照射。

 

使用说明

 

1. 推荐反应体系(推荐冰上配制)*****

组分	体积（μL）****	体积（μL）	终浓度
Hieff® qPCR SYBR Green Master Mix (High Rox Plus)*	25	10	1×
Forward Primer (10 μM)**	1	0.4	0.2 μM
Reverse Primer (10 μM)**	1	0.4	0.2 μM
模板 DNA***	X	X	–
无菌超纯水	to 50	to 20	–

* 使用前务必充分混匀，避免剧烈震荡产生过多气泡。

** 通常引物终浓度为0.2 μM，也可根据情况在0.1-1.0 μM之间进行调整。

*** 如模板类型为未稀释cDNA原液，使用体积不应超过qPCR反应总体积的1/10。cDNA原液建议5-10倍稀释，最佳模板加入量以扩增得到的CT值在20-30个循环为好。

**** 推荐使用20 μL或 50 μL，以保证目的基因扩增的有效性和重复性。

***** 请于超净工作台内配制，并使用无核酸酶残留的枪头、反应管；推荐使用带滤芯的枪头。避免交叉污染和气溶胶污染。

2. 反应程序

1. 两步法扩增程序*

循环步骤	温度	时间	循环数
预变性**	95℃	5 min	1
扩增反应	95℃	10 sec	40
	60℃***	30 sec****
熔解曲线	仪器默认设置		1

2. 三步法扩增程序

循环步骤	温度	时间	循环数
预变性**	95℃	5 min	1
扩增反应	95℃	10 sec	40
	55-60℃***	20 sec
	72℃	20 sec****
熔解曲线	仪器默认设置		1

* 高特异性可选择两步法，高效率扩增可选择三步法。

** 预变性时间可根据不同模板和引物的具体情况可适当缩短至2 min。

*** 退火温度和时间请根据引物和目的基因的长度进行调整。

**** 荧光信号采集请按照仪器使用说明书要求进行实验程序设置，几种常见仪器的时间设定如下：

30sec以上：Applied Biosystems: StepOne, StepOne Plus, 7500 Fast；Roche Applied Science: LightCycler 480；Bio-Rad: CFX96

31sec以上：Applied Biosystems: 7300

34sec以上：Applied Biosystems: 7500

3. 适用机型

Applied Biosystems: 5700, 7000, 7300, 7700, 7900HT FAST, StepOne, StepOne Plus.

4. 结果分析

定量实验至少需要三个生物学重复。反应结束后需要确认扩增曲线及熔解曲线。

1) 扩增曲线：标准扩增曲线为S型。

Ct值落在20-30之间时，定量分析最准确；

Ct值小于10，需要将稀释模板后，重新进行实验；

Ct值介于30-35之间时，需要提高模板浓度，或者增大反应体系的体积，以提高扩增效率，保证结果分析的准确性；

Ct值大于35时，检测结果无法定量分析基因的表达量，但可用于定性分析。

2) 熔解曲线：

熔解曲线单峰，表明反应特异性好可以进行定量结果分析；若熔解曲线出现双峰或者多峰，则不能进行定量分析。

熔解曲线出现双峰，需要通过DNA琼脂糖凝胶电泳判断非目标峰是引物二聚体还是非特异性扩增。

如果是引物二聚体，建议降低引物浓度，或者重新设计扩增效率高的引物。

如果是非特异性扩增，请提高退火温度，或者重新设计更高特异性的引物。

5. 引物设计指南

1）推荐引物长度25 bp左右。扩增产物长度150 bp为佳，可以在100 bp-300 bp内选择。

2）正向引物和反向引物的Tm值相差不宜超过2℃。引物Tm值60℃-65℃为佳。

3）引物碱基分布要均匀，避免出现连续的4个相同碱基，GC含量控制在50%左右。3’端最后一个碱基最好为G或C。

4）引物内部或者正反两条引物间最好避免出现有3个碱基以上的互补序列。

5）引物特异性需要用NCBI BLAST程序进行核对。避免引物3’端有2个碱基以上的非特异性互补。

6）设计完成的引物需要进行扩增效率的检测，只有具备相同扩增效率的引物才可用于定量比较分析。

 

注意事项

 

1. 本产品仅作科研用途。

2. 为了您的安全和健康，请穿实验服并佩戴一次性手套操作。

3. 解冻后Master Mix可能出现絮状物质，4℃放置并上下颠倒混匀至溶液澄清，不影响试剂性能。

4. 推荐使用本公司cDNA合成试剂盒（货号：11141ES），以有效去除RNA样品中残留的基因组。

 

Ver.CN20231116

 

Q：建议qPCR 实验用几步法？

A：常用 2 步法。需提高扩增特异性，可选用 2 步法或提高退火温度。在扩增效率低， ct 值过大的时候，可以改用 3 步法或延长延伸时间。

Q：预变性 5 min 调整成了 10min，对于实验结果有影响吗？

A：有影响，预变性时间较长可能会影响酶的活性，导致 PCR 产物的产量有所降低。

Q：qPCR 实验结果的有效性？为什么建议Ct 值要大于 15？

A：有效性要满足三个条件：（1）标准曲线：扩增效率范围:90–110%，对应斜率为  –3—3.5。 R2＞0.98。 (扩增效率=10-1/斜率–1)，当斜率=-3.32 时，扩增效率=100%。（2）扩 增曲线：S 型曲线，且 Ct 值在 15－35 之间，阴性对照 Ct＞35 或无 Ct 值。（3）熔解曲线：为单一峰。

Ct 值大于 15 个循环是因为 3-15 个循环的荧光值标准差的 10 倍是荧光阈值，Ct 值太小了会影响曲线。

Q：同一基因复孔间熔解曲线 Tm 值有差异？

A：同样的扩增产物也会出现Tm 值有微小差异，一般差异在 1 度以内都可以接受。

Q：为什么稀释了模板CT 值反而变小了？

A：一般 CT 值与模板起始浓度呈负相关，浓度越高，CT 值越小。但也有很多特殊情况， 比如体系中存在抑制物或是模板不纯，这时候稀释模板反而能使 CT 值变低。

Q：内参CT 值小于 20，目的基因均大于 30，怎么办？

A：可能是目的基因为低丰度表达基因导致。建议：a）换用内参；b）换引物；c）换检测线性范围更广的qPCR mix。

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