One Step SYBR Green RT-qPCR kit HYA417
One Step SYBR Green RT-qPCR Kit is a special kit for one-step RT-qPCR reaction by using SYBR Green I chimeric fluorescence. The kit takes RNA as the template, uses gene-specific primers, and reverse transcription and PCR reaction can be carried out continuously in the same tube without additional pipe opening and pipetting operations, greatly improving the detection flux and effectively preventing contamination. This reaction system can detect the amplification products in real time, greatly improving the detection sensitivity, and omitting the electrophoresis step after PCR reaction, which is very suitable for the detection of RNA virus and other trace RNA. This product integrates the superiority of Reverse Transcriptase and Taq DNA Polymerase, cooperate with optimized buffer system, with high amplification efficiency and high amplification specificity, one-step RT-qPCR reaction can be stable. In addition, all the enzymes used in the reaction are made into enzyme Mix, which is easier and more convenient to operate.
Components
|
Components |
25T |
250T |
|
2X One Step SYBR Green RT-qPCR Buffer* |
250 μL |
2 × 1.25 mL |
|
One Step Enzyme Mix** |
20 μL |
200 μL |
|
50X ROX Reference Dye I *** |
10 μL |
100 μL |
|
50X ROX Reference Dye II *** |
10 μL |
100 μL |
|
RNase-free ddH2O |
1.25 mL |
2 × 1.25 mL |
*contain dNTPs, Mg2+ , SYBR Green I, etc.
** contain Reverse Transcriptase, RNase Inhibitor , Taq DNA Polymerase.
*** Use to correct the error of fluorescence signal between holes
Storage
This product should be stored at -20℃ for long-term storage and should be protected from light.
Instruments
|
Rox types |
qPCR Machines |
|
No ROX |
Bio-Rad iCycler serious, Roche Light Cycler serious, Qiagen/Corbett serious and others |
|
ROX Reference Dye I |
ABI 7000/7300/7700/7900, ABI StepOne/StepOnePlus, Eppendorf and others |
|
ROX Reference Dye II |
ABI 7500, ABI ViiATM7, ABI QuantaStudio serious, Stratagene serious, Corbett Rotor Gene 3000 and others |
Note: The ROX Reference Dye is different for different instruments. For adding or not, please refer to the above models.
Notes
1. When using 2X One Step SYBR Green RT-qPCR Buffer, please fully melt it, mix it well and then use it. Avoid direct sunlight and keep it away from light. If multiple One Step RT-qPCR reactions need to be prepared at the same time, it is recommended to allocate all groups except primers and templates to make premix, and then separate it into each reaction tube to reduce the loss of reagents.
2. The One Step Enzyme Mix in the kit contains high concentration of glycerol, before using the Enzyme Mix, please mix gently and avoid foaming; Please use immediately after centrifugal. After usage please put it back -20℃ refrigerator.
3. The configuration and split charging of the reaction liquid must use non-polluting spear and Microtube to avoidcontamination as far as possible.
4. To ensure the success of the reaction, it is recommended to use high-quality RNA templates.
5. The kit can only use specific primers, and can't use random primers or Oligo dT primers for reverse transcription reaction.. When the one-step RT-qPCR experiment is designed for amplification of primers, the recommended product length of 70-200 bp has the best effect.
Instructions
1. qRT-PCR ReactionSystem
|
Components |
20uLVolume |
50uLVolume |
|
2X One Step SYBR Green RT-qPCR Buffer |
10uL |
25 µL |
|
One Step Enzyme Mix |
0.8 µL |
2 μL |
|
Forward primer (10 μM) * |
0.4 uL |
1 μL |
|
Reverse primer (10 μM)* |
0.4 uL |
1 μL |
|
50X ROX Reference Dye ** |
0.4 uL |
1 μL |
|
Template RNA *** |
2 μL |
5μL |
|
Nuclease-free H2O |
Up to 20 uL |
Up to 50 uL |
* The final concentration of primer is usually 0.2 μ M, which can get better results. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.1-1.0 μM. The length of the amplification product is recommended to be within the range of
70-200 bp.
**Select the appropriate ROX Dye according to the applicable model.
*** It is recommended to input 50 pg~300 ng Total RNA as template in 20 μL system.
2. RT-qPCR Reaction Program
|
Step |
Temperature |
Time |
Cycles |
|
Reverse Transcription |
42℃ |
5 min |
1 |
|
Pre-denaturation |
95 ℃ |
1 min |
1 |
|
Circular reaction |
95℃ |
5 s |
40 |
|
60℃ |
30-34 s * |
||
|
Melt Curve |
Instrument default |
- |
1 |
* Please adjust the extension time according to the minimum time limit for data collection required by your Real Time PCR device: set it to 30 s when using StepOnePlus; Please set it to 31 s when using 7300; Set it to 34 s when using 7500.
Confirm the amplification curve and melting curve after the reaction, make the standard curve.