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产品简介
Hieff® qPCR SYBR Green Master Mix(Low 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Low 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 |
11202ES03 / 11202ES08 / 11202ES50/ 11202ES60 |
|
规格 |
1 mL / 5×1 mL / 50×1 mL / 100×1 mL |
储存条件
-25~-15℃避光保存，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
- 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 (Low 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 |
- |
|
RNase Free H2O |
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，也可以根据情况在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总体积，以保证目的基因扩增的有效性和重复性。
- 扩增程序*
- 两步法
|
循环步骤 |
温度 |
时间 |
循环数 |
|
预变性** |
95℃ |
5 min |
1 |
|
变性 |
95℃ |
10 sec |
40 |
|
退火/延伸*** |
60℃ |
30 sec**** |
|
|
熔解曲线阶段***** |
仪器默认设置 |
1 |
|
2）三步法
|
循环步骤 |
温度 |
时间 |
循环数 |
|
预变性** |
95℃ |
5 min |
1 |
|
变性 |
95℃ |
10 sec |
40 |
|
退火*** |
55-60℃ |
20 sec |
|
|
延伸 |
72℃ |
20 sec**** |
|
|
熔解曲线阶段***** |
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
*****熔解曲线通常情况下可以使用仪器默认程序。
- 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
- 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
a. 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。
b. 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。
c. 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。
d. 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。
- 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。
5. 适用机型
Applied Biosystems: 7500, 7500 Fast, ViiA7, QuantStudio Dx, QuantStudio 3 and 5, QuantStudio 6,7,12k Flex;
Stratagene: MX3000P, MX3005P, MX4000.
注意事项
- 推荐使用本公司cDNA合成试剂盒（Cat#11141），以有效去除RNA样品中残留的基因组。
- 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。
- 为了您的安全和健康，请穿实验服并佩戴一次性手套操作。
- 本产品仅作科研用途。
Ver.CN20231128
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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