RT-LAMP Fluorescent Master Mix HYB413
This product contains reaction buffer, RT-enzymes mix of Bst DNA polymerase and heat-resistant reverse transcriptase, lyophilized protectant and fluorescent dye components. The reaction buffer contains Mg2+, dNTP and other necessary components for amplification, users can directly mix the reaction buffer, RT enzymes mix, fluorescent dye, primer, and then add the template. If necessary, users can add the lyophilized protectant to prepare lyophilizable system.
Components
|
Components |
96 T |
960 T |
9600 T |
|
Loop-mediated Amplification Buffer |
1.2 mL |
4 mL×3 |
12 mL×10 |
|
RT-Enzymes Mix |
245 μL |
2.45 mL |
2.45 mL×10 |
|
Lyophilized protectant |
0.96 mL×2 |
9.6 mL×2 |
9.6 mL×20 |
|
25× Dye |
192 μL |
0.96 mL×2 |
0.96 mL×20 |
Storage
Store at -25～-15℃, valid for 12 months.
Instructions
1. Thaw the reaction buffer to be used at room temperature. Vortex briefly or invert tubes several times to mix thoroughly, then centrifuge to collect the liquid to the bottom of the tube.
2. Preparation of reaction system: this reagent can be prepared in two reactionb systems, liquid reaction mix and Freeze- drying system mix.
1) Prepare liquid reaction mix
|
Components |
Volume |
|
Loop-mediated Amplification Buffer |
12.5 μL |
|
25× Dye |
2 μL |
|
RT-Enzymes Mix |
2.55 μL |
|
10×Primer Mix* |
5 μL |
|
Template DNA/RNA** |
x μL |
|
Nuclease-free Water |
Up to 50 μ L |
2) Freeze-drying system mix
① Prepare freeze-drying mix
|
Components |
Volume |
|
Loop-mediated Amplification Buffer |
12.5 μL |
|
Lyophilized protectant |
20 μL |
|
25× Dye |
2 μL |
|
RT-Enzymes Mix |
2.55 μL |
|
Nuclease-free Water |
Up to 50 μ L |
② Freeze dry
The prepared Mix was freeze-dried in a 50μL system.
③ Prepare reaction mix
|
Components |
Volume |
|
Freeze-dried mix |
1 piece |
|
10×Primer Mix* |
5 μ L |
|
Template DNA/ RNA** |
x μ L |
|
Nuclease-free Water |
Up to 50 μ L |
*10 × Primer Mix Concentration: 16 μMFIP/BIP, 2 μM F3/B3, 4 μM Loop F/B;
**DEPC (water soluble) is recommended for nucleic acid templ.
3. Reaction program
Set the parameters on the PCR instrument (such as ABI7500) as follows:
|
Temperature |
Time |
Cycles |
|
65℃ |
60s *Collect fluorescent signal |
30 |
Notes
1. Salt may appear in the bottom of the buffer tube, vortex briefly or invert tubes several times to mix thoroughly at room temperature.
2. The reaction temperature can be optimized between 62°C and 68°C according to the primer conditions.
3. The experiment shall be conducted in a standardized manner, including the preparation of reaction system, sample treatment and sample addition.
4. It is suggested to prepare reaction system in the ultra-clean table and add templates in the fume hood of other rooms to avoid false.