Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix是2×实时定量PCR扩增的预溶液，具有高灵敏度和高特异性的特点，颜色为蓝色，具有加样示踪的作用。核心组分Hieff UNICON® Taq DNA聚合酶采用抗体法热启动，可以有效抑制样品准备过程中引物退火导致的非特异性扩增。同时配方添加了提升PCR反应扩增效率因子和均衡不同GC含量（30~70%）基因扩增的促进因子，使定量PCR可以在宽广的定量区域内获得良好的线性关系。
本产品中含有特殊的ROX Passive Reference Dye，适用于所有qPCR仪器，无需在不同的仪器上调整ROX的浓度，只需在配制反应体系时加入引物和模板即可进行扩增。
- 全仪器平台通用：预混特定类型参比染料，无需调整ROX浓度
- 易于示踪：蓝色预混液，观察颜色可直接区分是否加样
- 强稳定性：室温配制体系，配制后的体系可4℃放置24 h，检测结果无变化
- 上机快速：兼容常规程序与快速程序，最快46 min完成定量实验
- 扩增性能好：扩增效率高，特异性好，可检测个位拷贝数基因
- 全平台通用：适用于所有qPCR机型
图1. Hieff UNICON® Universal Blue 预混液可实现全平台通用。针对不同仪器平台，与T品牌对比，Hieff UNICON® Universal Blue 预混液Ct值起峰更加靠前。以2 µL的质粒10倍梯度稀释液为模板，扩增IL23R基因。
- 灵敏度高：可检测至单拷贝
图2. Hieff UNICON® Universal Blue 预混液可有效检测7个数量级范围的模板量，扩增效率高，可在宽广的线性范围内获得良好的线性关系。以2 µL的106-100拷贝的质粒为模板，扩增人IL23R基因。
- 检出率和特异性良好：广泛适用于30-70%GC含量的扩增子
图3. Hieff UNICON® Universal Blue预混液广泛适用于30-70%GC含量的扩增子，保证了极高的特异性检出率。以2 µL的293T cDNA为模板，利用Primer5软件设计1000对长度为200 bp扩增子（GC%含量30%-70%）的引物，随机扩增其中的27个扩增子。
- 分辨率高：可精准分辨2倍模板浓度差异
图4. Hieff UNICON® Universal Blue 预混液可精准区分2倍模板浓度差异。以2 µL的293T cDNA原液的2倍梯度稀释液为模板，扩增GAPDH基因。
- 复孔重复性好：90个复孔
图5. Hieff UNICON® Universal Blue预混液具备良好的重复性，90个复孔的扩增曲线高度重合，Ct值标准偏差<0.2。以1 µL的293T cDNA为模板，扩增GAPDH基因。
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。
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：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：11184 的灵敏度极限是?
A：单拷贝
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
Q：为什么扩增曲线杂乱且不连续？
A：可能原因是 ROX 添加不当。确认参比染料ROX 是否添加正确。
Q：模板用量X 是多少？常用的量是多少？
A：(1)X 表示模板 DNA 量需要实验者在首次实验时进行摸索。首先对模板DNA 进行稀释（一般推荐 5-10 倍），然后模板量梯度上样，选择 CT 值落在 20-30 之间的最佳上样量。
(2)常用的量是逆转录 500-1000ng 总RNA，稀释 10 倍取 1μL cDNA 进行qPCR 实验。
Q：qPCR 实验结果的有效性？为什么建议Ct 值要大于 15？
A：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。
b)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：Rox 的作用？
A:ROX 是一种参比染料，其作用是标准化荧光定量反应中的非PCR 震荡，校正加样误差或者是孔与孔之间的误差，提供一个稳定的基线。
Q: 为什么扩增产物跑胶会有拖尾的现象？
A: 试剂中含有稳定性成分，本身不参与任何反应，但在电泳胶上面会表现出弥散状，不建议跑完后再电泳。
Q: 试剂比较粘稠，容易产生气泡，如何避免？
A: 可以先混合大体系，再分别加到离心管里面，最后加模板；
枪头加样不要把空气打进去，二档吸样，一档打样品；缓慢推出液体，不要吹打；沿着壁打入到孔内；
另外配置完后，如果还有气泡，离心PCR管去除。如果是96孔板，移液完后轻轻敲96孔板。
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