Direct RT-qPCR Kit II
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,high efficient Foreasy HS Taq DNA Polymerase and Foreasy Reverse Transcriptase(MMLV), making it convenient for the creating of back-end kits and system adjustment, within simple verification.
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.
This kit can directly be the component of the IVD product, using easily and fast. It only needs easy verification, without developing again.
Specifications
500T, 50,000T
Kit components
| Kit contents
(20 μL reaction system) |
IM-05111-02 | IM-05112-02 |
| 500 T | 50,000 T | |
|
Foreasy Reverse Transcriptase (200 U/μL) |
50 μL ×1 | 1 mL ×5 |
|
Foreasy HS Taq DNA Polymerase (5 U/μL) |
100 μL ×1 | 1 mL ×10 |
| 2× Direct RT-qPCR Buffer | 1 mL ×5 | 500 mL ×1 |
| Instruction | 1 | 1 |
Operating Steps
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
Get half the volume of reaction buffer of reaction system (for example, if the volume of the system is 20 μL, it needs to get 10 μL 2× Direct RT-qPCR Buffer). For corresponding amount of enzyme,we suggest that M-MLV：10-30 U/20 μL reaction system,Taq DNA polymerase：1-2 U/20 μL reaction system this is our suggestion,you should test and adjust it), add RNA template, specific primers and probes, and add RNase-Free ddH2O to 20 μL. The specific preparation of RT-qPCR reaction system can be referred to the following table 1.
Table 1 : Prepare of RT-qPCR system
| Component | Volume | Final concentration |
| 2× Direct RT-qPCR Buffer | 10 μL | 1× |
| Foreasy Reverse Transcriptase(MMLV) | 10-30 U | |
| Foreasy HS Taq DNA Polymerase | 1-2 U | |
| 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 |
Note: Forward Primer and Reverse Primer are specific primers of target gene. The system of the qPCR could be adjusted according to practical experimental and PCR models. We recommend 400nM for the final concentration of most primers. Adjust the volume of specific primers and probes according to the prepared concentration and the recommended final concentration, please.
1*: Select suitable final concentration of ROX Reference Dye according to different quantitative PCR instruments. The optimal ROX Reference Dye concentration of common quantitative PCR instruments is shown in the table following:
| Quantitative PCR instruments | Final concentration of ROX Reference Dye |
| ABI PRISM 7000/7300/7700/7900HT/Step One,etc. | 1× (for example, add 1μl 20×ROX Reference Dye in 20 μL reaction system) |
| ABI 7500, 7500 Fast, Stratagene Mx3000P, Mx3005P and Mx4000, etc. | 0.5× (for example, add 0.5μl 20×ROX Reference Dye in 20 μL reaction system) |
| Roche PCR instrument, Bio-Rad PCR instrument, Eppendorf quantitative PCR instrument, etc. | Without ROX Reference Dye |
Storage and Shelf life
The kit should be stored at -20 ± 5℃. It should immediately be stored in -20℃ thermostatic refrigerator. The validity period is 2 years if it be stored in suitable condition.
