RT-LAMP Colorimetric Master Mix HCB5204A
This product contains reaction buffer, RT-Enzymes Mix (Bst DNA polymerase and heat-resistant reverse transcriptase), lyophilized Protectants and chromogenic dye components. To use, just use Buffer, the reaction enzyme and primer are mixed and added to the template; adding lyophilized protectant can be straight. It was connected to a lyophilizer and lyophilized, and only the primers and templates were added when used. This kit provides a fast, clear visual detection of amplification, which negative reaction is indicated in red and positive reaction is indicated by a change to yellow.
Component
|
Component |
HCB5204A-01 |
HCB5204A-02 |
HCB5204A-03 |
|
Loop-mediated Amplification Buffer (with dye) |
0.96 mL |
4.80 mL×2 |
9.60 mL×10 |
|
RT-Enzymes Mix |
270 μL |
2.70 mL |
2.70 mL×10 |
|
Lyophilized protectant |
0.96 mL×2 |
9.60 mL×2 |
9.60 mL×20 |
Applications
For DNA or RNA isothermal amplification.
Storage Conditions
Transported with Dry ice, stored at -25~ -15℃. Avoid frequent freeze-thaw, the product is valid for 12 months.
Protocol
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 reaction systems, liquid reaction mix and lyophilized system mix.
1) Prepare liquid reaction mix
|
Component |
Volume |
|
Loop-mediated Amplification Buffer (with dye) |
10 μL |
|
RT-Enzymes Mix |
2.8 μL |
|
10 × Primer Mix a |
5 μL |
|
Templates DNA/ RNA b |
× μL |
|
Nuclease-free Water |
Up to 50 μL |
2) Lyophilization system mix
① Prepare lyophilized mix
|
Component |
Volume |
|
Loop-mediated Amplification Buffer (with dye) |
10 μL |
|
Lyophilized protectant |
20 μL |
|
RT-Enzymes Mix |
2.8 μL |
|
Nuclease-free Water |
Up to 50 μL |
② Lyophilization: The prepared Mix was lyophilized in a 50μL system
③ Prepare reaction mix
|
Component |
Volume |
|
Lyophilized mix |
1 piece |
|
10 × Primer Mix a |
5 μL |
|
Templates DNA/ RNA b |
× μL |
|
Nuclease-free Water |
Up to 50 μL |
Notes:
1) a. 10×Primer Mix : 16 μM FIP/BIP, 2 μM F3/B3, 4 μM Loop F/B;
2) b. DEPC (water soluble) is recommended for nucleic acid templ.
1.Incubate at 65°C for 30-45mins, which can be extended appropriately according to color change Reaction time.
2.According to the naked eye, yellow was positive and red was negative.
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 ℃ and 68 ℃ according to the condition of primers.
3.The packaged reagents should not be exposed to air for a long time.
4.The red and yellow discoloration reaction depends on the pH change of the reaction system, please do not use the containing Tris nucleic acid storage solution, recommended to use ddH2O stored nucleic acid;
5.The experiment should be standardized, including the preparation of the reaction system, lyophilization, and sample processing and sample adding process;
6.To avoid contamination, it is recommended to prepare the reaction system in an ultra-clean bench, in other Add templates to the fume hood of the room to avoid false positive interference.
