Direct RT-qPCR Kit
Kit Description:
◮ Direct RT-qPCR Kit, using Foreasy Reverse Transcriptase and Foreasy HS Taq DNA Polymerase developing by Foregene, with unique reaction buffer, makes this kit with strong resistance and compatibility, and it can directly test reaction use Foregene Lysis system as template. This kit can apply for COVID-19 nucleic acid detection kit and other pathogenic bacteria nucleic acid detection kit development.
Description
Direct RT-qPCR Kit provides separately packaged RT-qPCR buffer and high efficient enzyme mixture(Taq、M-MLV、RI)，making it convenient for the creating of back-end kits and system adjustment，within simple verification.
Specifications
500T, 50,000T
Kit components
| Kit contents
(20 μL reaction system) |
IM-05111-01 | IM-05112-01 |
| 500 T | 50,000 T | |
| Direct Enzyme Mixture | 500 μL ×1 | 50 mL ×1 |
| 2× Direct RT-qPCR Buffer | 1 mL ×5 | 500 mL ×1 |
| Instruction | 1 | 1 |
Operating Steps
Before reagent preparation, all the reagents in the kit should be thawed at room temperature and mixed gently.
A: Prepare of template and reagent
1. Prepare prepared RNA template (It is recommended to use Foregene Total RNA Isolation Kit series kit to extract and purify RNA template) or sample cracking product (It is recommended to use Foregene Lysis system), specific primers(10 μM) and other relevant consumables, instrument.
2. Put the 2× Direct RT-qPCR Buffer, RNase-Free ddH2O and 20× ROX Reference Dye(if it’s needed) in the box with ice, making these melt naturally, and mixed gently.
B: Prepare of RT-qPCR system
Table 1 : Prepare of RT-qPCR system
| Component | Volume | Final concentration |
| 2× Direct RT-qPCR Buffer | 10 μL | 1× |
| Direct Enzyme Mixture | 1 μL | |
| Forward Primer(10 μM) | 0.8 μL | 50-900nM |
| Reverse Primer(10 μM) | 0.8 μL | 50-900nM |
| Probe(4 μM) | 1 μL | 200nM |
| Template(RNA or Lysate) | X μL | |
| 20×ROX Reference Dye | - | 1* |
| RNase-FreeddH2O | (6.4-X) μL | |
| Total Volume | 20 μL |
C: Set the RT-qPCR reaction program
Table 2 : RT-qPCR reaction program setting (for example: two-step method)
| Step | Temperature | Time | Cycles | Content | |
| 1 | 50℃ | 15 min | 1* | 1 | Reverse transcription |
| 2 | 95℃ | 1 min | 1 | Pre-denaturation | |
| 3 | 95℃ | 10 sec | 40-45 | Denaturation | |
| 60℃ | 30 sec 2* | Anneal/Extension | |||
Storage and Shelf life
-The kit should be stored at -20 ± 5℃. The validity period is 12 months.
-Avoid repeated freeze-thaw cycles (<5 cycles).
