Store at −20°C for long-term storage and at 4°C for short-term use. Protect from light.
ProtoGreen Fast qPCR Mix
Product Components
| Components | Component Number | Size-1 (5 mL) | Size-2 (25 mL) |
|---|---|---|---|
| ProtoGreen Fast qPCR Mix* | MZ21204 | 5 × 1 mL | 25 × 1 mL |
| 50X ROX Reference Dye I | MZ21465 | 200 µL | 5 × 220 µL |
| 50X ROX Reference Dye II | MZ21466 | 200 µL | 5 × 200 µL |
* Including Molzyme Hot Start Taq DNA Polymerase, Mg²⁺, dNTPs, SYBR® Green, etc.
Product Description
Real-time quantitative PCR (qPCR) is a technique that uses double-stranded DNA-binding dyes, usually SYBR® Green, to quantitatively detect the initial amount of DNA during amplification.
Molzyme ProtoGreen Fast qPCR Mix is a fluorescence reagent designed for qPCR reactions using SYBR® Green chemistry. This product provides real-time data on DNA amplification during PCR by detecting quantitative fluorescence signals in the SYBR®/FAM channel.
The mix utilizes an antibody-based hot-start Taq DNA polymerase, which significantly improves amplification specificity while maintaining high efficiency. In addition, the optimized qPCR buffer system makes the product suitable for multiple species, providing a powerful tool for multidisciplinary experimental applications.
This product is supplied as a 2X premixed enzyme solution containing all components required for qPCR except primers and template DNA, offering maximum convenience for experimental setup.
Instruments
| ROX Type | Compatible qPCR Machines |
|---|---|
| No ROX | Bio-Rad iCycler series; Roche LightCycler series; Qiagen/Corbett series; and others |
| ROX Reference Dye I | ABI 7000 / 7300 / 7700 / 7900; ABI StepOne™ / StepOnePlus™; Eppendorf systems; and others |
| ROX Reference Dye II | ABI 7500; ABI ViiA™ 7; ABI QuantStudio™ series; Stratagene series; Corbett Rotor-Gene 3000; and others |
Note: The requirement for ROX Reference Dye varies depending on the instrument. Please refer to the compatible models listed above to determine whether ROX should be added and which type to use.
Storage
Materials Required
EP tubes, PCR tubes, and other related consumables
qPCR-specific primers and templates
qPCR plates and sealing membranes
Usage Notes
Before using ProtoGreen Fast qPCR Mix, ensure the mix is completely thawed, then place it on ice before use.
Mix the qPCR components thoroughly and gently with the ProtoGreen Fast qPCR Mix by pipetting or brief vortexing. After use, store the mix at −20°C for long-term storage or 4°C for short-term use.
Since ProtoGreen Fast qPCR Mix contains Taq DNA polymerase, all operations should be performed on ice.
ProtoGreen Fast qPCR Mix is compatible with ROX reference dye. Add the appropriate ROX dye according to the qPCR instrument model being used.
To avoid contamination, use pipette tips with filters.
For optimal qPCR results, use high-quality DNA templates.
Protocol
Before Use
Check the specificity of primers. A final primer concentration of 0.2 µM is suitable for most applications.
The length of amplification products is usually in the range of 70–200 bp.
Dilute the template DNA in a gradient to determine the optimal concentration.
Add 1 pg–50 ng DNA as the PCR template. A No Template Control (NTC) sample is recommended.
To ensure experimental reliability, perform at least two replicates for each sample.
Experimental Procedure
Prepare the following reaction systems on ice:
| Component | Volume |
|---|---|
| ProtoGreen Fast qPCR Mix (2X) | 10 µL |
| Forward Primer (10 µM) | 0.4 µL |
| Reverse Primer (10 µM) | 0.4 µL |
| gDNA or cDNA (<50 ng) | 2 µL |
| ROX Reference Dye | 0.4 µL |
| ddH₂O | To 20 µL |
- Dissolve ProtoGreen Fast qPCR Mix at room temperature, then place it on ice before use. Prior to use, mix thoroughly by gentle inversion or vortexing, and briefly centrifuge to collect the solution at the bottom of the tube.
- Calculate the total volume of reaction mix required. It is recommended to prepare an additional 10% excess volume to compensate for pipetting loss.
- Dispense the solution into sterile PCR tubes or EP tubes to avoid contamination.
- Add all components listed in the reaction table above. Mix gently but thoroughly (avoid bubble formation), then briefly centrifuge to collect the contents.
- Dispense the reaction mixture into qPCR plates and seal the plates with an optical sealing membrane.
- Centrifuge the qPCR plates at 2,500 rpm to collect all the solution at the bottom of the wells before placing them into the instrument.
2.Program qPCR reactions follows:
| Stage | Step | Repetitions | Temperature | Time |
|---|---|---|---|---|
| Stage 1 | Initial Denaturation | 1 | 95°C | 3 min |
| Stage 2 | Denaturation | 40–45 cycles | 95°C | 5 s |
| Annealing/Extension* | 60°C | 30–34 s | ||
| Stage 3 | Melt Curve | 1 | Default instrument settings | — |
* Confirm that fluorescence signal collection is performed after each extension step. The extension time may vary depending on the instrument: 30 s for StepOnePlus™, 31 s for ABI 7300, and 34 s for ABI 7500.
Draw a standard curve based on the Ct values of the endogenous gene. The R² value should be greater than 0.98, and the slope should be between −3.0 and −3.5, indicating a PCR amplification efficiency between 90% and 120%.
The standard deviation (SD) of Ct values should be less than 0.2, and the variation in Ct values between different experiments should be less than 0.5. When comparing Ct values across experiments, ensure that the threshold settings remain consistent.
A single peak in the melt curve indicates the absence of non-specific amplification products or primer dimers. The Tm value in the melt curve is typically in the range of 80–95°C.
Troubleshooting
Melt Curve Shows Multiple Peaks
a. Improper Primer Design:
Design primers according to standard primer design guidelines to ensure specificity.
b. Primer Concentration Too High:
Reduce the primer concentration to minimize non-specific amplification.
Unusual Amplification Curves
a. Amplification Curve Not Smooth: Signal may be too low. Increase template input and ensure the qPCR Mix is stored properly.
b. Inconsistent Amplification Curve: Bubbles may cause abnormal results. Centrifuge the plate prior to running.
c. Abnormal Amplification Curves: The default baseline setting of the machine is usually 3–15 cycles. Adjust the baseline according to actual amplification conditions. Template degradation may also affect the curve.
No Amplification Curves After Reaction
a. Not Enough PCR Cycles: The PCR cycle number is usually set to 40. A higher cycle number may increase background signal.
b. Primer Degradation: Use electrophoresis to confirm primer integrity.
c. Confirm Signal Collection Step: For two-step qPCR, signal collection is usually set after the annealing/extension step. For three-step qPCR, signal collection is usually set after the extension step.
d. Template Input Too Low: Increase template concentration or add extra replicates.
e. Template Degradation: Use freshly prepared template. Confirm integrity by electrophoresis.
f. Not Enough Initial Denaturation Time: ProtoGreen Fast qPCR Mix uses Hot-Start Taq polymerase; pre-denaturation time should be at least 3 min.
Ct Value Too Late
a. Low Amplification Efficiency: Optimize reaction conditions or change primers.
b. Template Input Too Low: Increase template concentration or repeat the experiment.
c. Template Degradation: Use freshly prepared template and confirm integrity by electrophoresis.
d. PCR Product Too Long: Amplification product length is usually 70–200 bp.
e. PCR Inhibition Reagents: Use new template or dilute the template.
f. Pre-denaturation Time Too Short: Pre-denaturation time should be at least 3 min.
NTC Shows Amplification
a. Contamination: Use sterile water and perform all operations in a clean room to avoid aerosol contamination.
b. Non-Specific PCR Products: Analyze with melt curve.
Inconsistent Results
a. Inconsistent Sample Addition: Use proper pipetting techniques.
b. Inconsistent Temperature in qPCR Machine: Ensure periodic machine calibration.
c. Template Concentration Too Low: Increase template concentration.
d. Inconsistent Threshold Setting: When comparing results from different plates, ensure the threshold value is the same across experiments.