RT-LAMP Probe Master Mix HYB415
This product contains reaction buffer, Bst DNA polymerase and RT-Enzymes Mix of thermostable reverse transcriptase, and freeze-dried protective agent components. The reaction buffer contains Mg2+, dNTP and other necessary components for amplification. When using, you only need to mix the buffer, reaction enzyme, primer probe and add the template; it can also be used to prepare a freeze-dried reaction system. After adding the freeze-dried protective agent, it can be directly freeze-dried in a freeze dryer. When using, you only need to add the primer probe and template. This reagent has high amplification efficiency and sensitivity.
Components
|
Components |
96 T |
960 T |
9600 T |
|
Loop-mediated Amplification Buffer |
1 mL |
2.5 mL×4 |
10 mL×10 |
|
RT-Enzymes Mix |
360 μL |
3.6 mL |
3.6 mL×10 |
|
Lyoprotectant |
1 mL |
2.5 mL×4 |
10 mL×10 |
Storage
Store at -25～-15℃,valid for 12 months
Instructions
1. Take out the buffer and thaw it. Vortex thoroughly before use, and centrifuge briefly to collect the liquid at the bottom of the tube.
2. Preparation of reaction system：This reagent has two types of reaction systems: liquid reaction system and freeze-dried reaction system.Please choose according to the specific usage.
1) Preparation of liquid reaction system
|
Component |
Volume |
|
Loop-mediated Amplification Buffer |
10 uL |
|
RT-Enzymes Mix |
3.6 μL |
|
10×Primer probe Mix* |
5 μL |
|
Template RNA** |
X μL |
|
Nuclease-free Water |
Up to 50 μL |
2) Preparation of freeze-dried reaction system
① Preparation of freeze-dried system
|
Component |
Volume |
|
Loop-mediated Amplification Buffer |
10 uL |
|
Lyoprotectant |
10 uL |
|
RT-Enzymes Mix |
3.6 μL |
|
Nuclease-free Water |
Up to 25 μL |
② Freeze-drying
Freeze-dry the prepared Mix according to the 25μL system.
③ Preparation of reaction system
|
Component |
Volume |
|
Freeze-dried products |
1 Piece |
|
10×Primer probe Mix* |
5 μL |
|
Template RNA** |
x μL |
|
Nuclease-free Water |
Up to 50 μL |
*10×Primer Mix concentration: 16 μM FIP/BIP, 2 μM F3/B3, 4 μM Loop F/B，4 μM probe;
**It is recommended to dissolve the nucleic acid template in DEPC water.
3. Amplification reaction
Set up the following program on a fluorescent PCR instrument (such as ABI7500, etc.).
|
Temperature |
Time |
Cycles |
|
65℃ |
60s *Collect FAM fluorescence |
30 |
Notes
1. After the buffer is melted, there may be white precipitate, which can be clarified by mixing thoroughly at room temperature;
2. The reaction temperature can be optimized between 62℃-68℃ according to the primer situation;
3. The experiment should be operated in a standardized manner, including the preparation of the reaction system, freeze-drying, sample processing and sample addition.