5X One Step SYBR Green RT-qPCR Premix HYA415
One Step SYBR Green RT-qPCR Premix is for use with SYBR Green Ⅰ for Real Time PCR. This product is used in one step (One Step) RT-PCR fluorescent quantitative method, counter-current process within the same jurisdiction, avoidance development, effective effect prevention and contamination. This product contains and removes RNaseH active high-temperature reaction time RTase with a new type of heat reaction time, has higher reaction time PCR increase efficiency, and is suitable for low concentration RNA model high sensitivity increase. This study has a special arrangement, a better arrangement for qPCR, and a better Buffer for qPCR. This review has been provided by many manufacturers of fluorescent quantitative PCR, such as AppliedBiosystems, Eppendorf, Bio-Rad, Roche, etc.
Components
1. 25×RTase Mix (SYBR qRT-PCR)
2. 5× RT Premix Buffer (SYBR qRT-PCR)
Storage
This product should be stored away from light at -20℃ for long-term storage and 4℃ for 3 months.
Instructions
1. qRT-PCR ReactionSystem
|
Components |
25 uL Volume |
50 uL Volume |
Final Concentration |
|
5 × RT Premix Buffer (SYBR qRT-PCR) |
5 uL |
10 uL |
1× |
|
25× RTase Mix (SYBR qRT-PCR) |
1 uL |
2 uL |
1× |
|
25×Primer Mix* |
1 uL |
2 uL |
1× |
|
RNA template** |
- |
- |
- |
|
DEPC H2O |
Up to 25 uL |
Up to 50 uL |
- |
*Usually, a final primer concentration of 0.2 μM can produce good results; when the reaction performance is poor, the primer concentration can be adjusted within the range of 0.2-1 μM.
**The recommended amount of RNA template to be added is between 10 pg and 100 ng.
2. qRT-PCR Reaction Program
|
Step |
Temperature |
Time |
Cycles |
|
Reverse transcription |
50 ℃ |
10-20 min |
1 |
|
Pre Denaturation |
95 ℃ |
5-10 min |
1 |
|
Denaturation |
95 ℃ |
10-20 s |
40-50 |
|
Annealing and Extension |
50-64℃ |
20-60 s |
|
|
Melt Curve |
Instrument default |
/ |
1 |
* Confirm there is a signal collection step after each extending step. The extending time is varied according to different machines: 30 s for StepOne Plus, 31 s for 7300 and 34 s for 7500. If not otherwise specified, default to 30 s.
Technical Information
1. The system is usually used at 50℃, the temperature can be improved at 42℃~55℃, and the temperature can be improved within 5-30 minutes.
2. The RTase used in the text system is the M-MLV basic modification, has higher temperature tolerance and higher reversal temperature, and has higher reversal efficiency.
3. The system has a good system definition, compatibility, very suitable for pathogenicity, and the organization of RNA. Add a model to the test; use a low-strength model to increase its effectiveness further, and then use a high-sensitivity molecular test.
4. The three-step method for increasing the extinguishing temperature to lower or exceed 200 bp length.
5. Before and after use, the liquid transfer device should be used, and the operation procedure of the PCR tube should be constantly updated; after the completion of the PCR reaction, the pipe should not be opened, so as to reduce the soil contamination to the maximum extent possible.