Using different amounts of HEK293T total RNA as templates, the DNA fragment of 197bp of the β-actin gene was detected. The electrophoresis results of RT-PCR amplification after adding a specified amount of this product or similar products from Company T (competitor) in a 50μl reaction system are shown in the figure. As shown in the figure, this product has a consistent detection sensitivity compared to Company T's products, and can sensitively detect RNA as low as 1pg, and has better specificity and amplification efficiency.
M: DL2000 DNA Marker；
CK: No Enzyme Mix added；
Lane 1: 1ug；
Lane 2: 100ng；
Lane 3: 10ng；
Lane 4: 1 ng；
Lane 5: 100pg；
Lane 6: 10pg；
Lane 7: 1pg；
Lane 8: 0.1 pg；
StartScript® One Step RT-PCR Kit
StartScript® One Step RT-PCR Kit
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$90 USD
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$90 USD
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Product Details
Product Details
Product Specification
| Stability & Storage | Store at -25 ~ -15℃ for 2 years |
| Reference |
Components
Component |
UA070126-50 Rxns |
UA070126-250 Rxns |
One-Step Enzyme Mix (25X) |
100 μl |
5 × 100 μl |
One-Step Reaction Mix (2X) |
2 × 625 μl |
10 × 625 μl |
RNase Free dH2O |
2 × 1 ml |
10 × 1 ml |
Protocol
1. 在Rnase-free离心管中配制如下混合液：
Component |
Volume |
One-Step Enzyme Mix (25X) |
2 μl |
One-Step Reaction Mix (2X) |
25 μl |
Gene-specific Forward Primer (10 μM) |
2 μl |
Gene-specific Reverse Primer (10 μM) |
2 μl |
Template RNA (1 pg~1 μg) |
x μl |
Rnase Free dH2O |
Up to 50 μl |
用移液器轻轻吹打混匀。
2. 按下列条件进行RT-PCR反应
Step |
Temperature |
Time |
Number of Cycles |
Reverse Transcription* |
48°C |
30 minutes |
1 cycle |
Initial Denaturation |
94°C |
3 minutes |
1 cycle |
|
Denaturation Annealing** Extension |
94°C 50-65°C 72°C |
30 seconds 30 seconds 1 minutes |
25–35 cycles
|
Final Extension |
72°C |
5 minutes |
1 cycle |
Soak |
4°C |
Indefinite |
1 cycle |
*如果模板具有复杂二级结构或高GC区域，可将反应温度提高至55℃，有助于提高产量。
**退火温度需要根据引物退火温度调整，一般设置成低于引物退火温度1 ~ 2℃即可。
Guidelines
1. 使用DEPC处理研究中使用的所有设备，或购买经证明无核酸的设备。在研究过程中戴上手套，并经常更换，以避免RNA酶污染。
2. 确保所用试剂中没有RNA酶污染。
3. 通常情况，28～30个循环可达到最佳扩增，对于低拷贝目的基因的检测，可将循环数增加到40个。
4. 使用本试剂盒进行反转录反应时必须使用特异性的反转录引物，Random Primer、Oligo dT Primer不能使用。
5. 实验操作时应将酶制品放在冰上，实验结束后立即保存在 -20°C。
Picture
Picture
Bioactivity
