产品说明书
FAQ
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已发表文献
产品描述
Hieff® qPCR SYBR Green Master Mix(No Rox)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+。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。
适用机型
Bio-Rad: CFX96, CFX384, iCycler iQ, iQ5, MyiQ, MiniOpticon, Opticon, Opticon 2, Chromo4;
Eppendorf: Mastercycler ep realplex, realplex 2 s;
Qiagen: Corbett Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000;
Roche Applied Science: LightCycler 480, LightCycler 2.0; Lightcycler 96;
Thermo Scientific: PikoReal Cycler; Cepheid: SmartCycler; Illumina: Eco qPCR.
运输和保存方式
冰袋运输。-20℃避光储存，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。
注意事项
1. 推荐使用本公司cDNA合成试剂盒（货号：11123ES），以有效去除RNA样品中残留的基因组。
2. 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。
3. 为了您的安全和健康，请穿实验服并佩戴一次性手套操作。
4. 本产品仅作科研用途！
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 (No Rox) |
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 |
- |
【注】： 使用前务必充分混匀，避免剧烈震荡产生过多气泡。
a) 引物浓度：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。
b) 模板浓度：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。
c) 模板稀释：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。
d) 反应体系：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。
e) 体系配制：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。
【注】: 高特异性可选择两步法，高效率扩增可选择三步法。
a) 预变性时间：根据不同模板和引物的具体情况可适当缩短至2 min。
b) 退火温度和时间：请根据引物和目的基因的长度进行调整。
c) 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
d) 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：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时，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。
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。
相关产品
|
产品名称 |
货号 |
规格 |
|
11119ES60 |
100 T |
|
|
Hifair® II 1st Strand cDNA Synthesis Kit (gDNA digester plus) |
11121ES60 |
100 T |
|
Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR(gDNA digester plus) HOT |
11123ES60 |
100 T |
|
11201ES08 |
5 ml |
|
|
11202ES08 |
5 ml |
|
|
11203ES08 |
5 ml |
HB210720
Q：不加 ROX 的qPCR mix 后续需换用其他仪器（需要 ROX），是否可以单独加 ROX后再使用？
A：可以。
Q：可以用于microRNA 或lncRNA 等非编码RNA 吗？环状 RNA 呢？
A：不推荐。
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：11201,11202,11203区别是什么呢？如何选择呢？
A：里面预混的ROX浓度不同，需要根据仪器的型号进行对应选择。
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