SYBR Green qPCR Premix HYA412
Real-time Quantitative PCR (qPCR) is a powerful technique in detecting initial DNA input in a PCR reaction by fluorescence signal accumulation. The DNA double strand bonded dye, SYBR® Green I is the most commonly used dye in qPCR. SYBR Green qPCR Premix contains de novel designed universal reference dye, which can realize higher signal resolution and suits for all currently used qPCR instruments (including high ROX mode, low ROX mode and No ROX mode required machines). It also contains Hot-Start Taq DNA polymerase to avoid unexpected amplification results. Besides, SYBR Green qPCR Premix is an optimized qPCR reaction mix, It contains all required components in qPCR except primers and template. It is convenient for experiment and suitable for multiple species. The above features make it as an ideal experiment tool for gene quantitative research.
Components
|
Components |
1 mL |
5 mL |
25 mL |
|
SYBR Green qPCR Premix* |
1mL |
5 × 1mL |
25 × 1mL |
*Including Hot Start Taq DNA polymerase, Mg2+, dNTPs, SYBR® Green I, Universal ROX Dye，etc.
Instruments
No additional reference dye is required. SYBR Green qPCR Premix is suited for all currently used qPCR instruments (including high ROX mode, low ROX mode and No ROX mode required machine).
Storage
This product should be stored at -20℃ for long-term storage and should be protected from light.
Materials Required
1. PCR tubes and other related materials.
2. qPCR specific primers and DNA templates.
3. qPCR 96-well plate and sealing membrane (adhesive film).
Notes
1. SYBR Green qPCR Premixmust be completely thawed before use. Protect from direct light exposure and store in darkness.
2. SYBR Green qPCR Premixcontains glycerol. Gently mix before use to avoid bubble formation; vortex and centrifuge before use. Immediately return to -20°C storage after use.
3. This product contains DNA polymerase. Maintain on ice throughout use. For short-term repeated usage within a day, temporary storage at 4°C is permitted. Minimize freeze-thaw cycles.
Instructions
1. Experimental Preparation
1. It is recommended to choose the amplification product length within the range of 70-200 bp.
2. It is recommended to take a reaction volume of 20 μL, add 1 pg-50 ng of DNA as a template, and set NTC (No Template Control).
3. To ensure experimental accuracy, perform triplicate technical replicates for all samples and controls.
2. qPCR ReactionSystem
|
Components |
Volume |
|
SYBR Green qPCR Premix |
10uL |
|
Forward Primer (10 µM)* |
0.4 uL |
|
Reverse Primer(10μM)* |
0.4 uL |
|
gDNA or cDNA** |
2uL |
|
ddH2O |
Up to 20 uL |
*Typically, the final concentration of the primer is 0.2 μM，and good results can be obtained , and the final concentration of 0.1-1.0 μM can be used as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the concentration of primers can be reduced and the reaction system can be optimized.
**Using 10 pg-10 ng genomic DNA or 10 pg-100 ng cDNA as the template reference quantity, gradient dilution can be performed on the template to determine the optimal template usage due to the different copy numbers of the target genes contained in the templates of different species. In addition, when using cDNA (RT reaction solution) from the two-step RT qPCR reaction as a template, the addition amount should not exceed 10% of the qPCR reaction system.
3. qPCR Reaction Program
|
Step |
Temperature |
Time |
Cycles |
|
Pre Denaturation |
95 ℃ |
3 min |
1 |
|
Denaturation |
95 ℃ |
5 s |
40-45 |
|
Annealing and Extension |
60℃ |
30-34 s* |
|
|
Melt Curve |
Instrument default |
/ |
1 |
* Confirm there is a signal collection step after each extending step. The extending time is varied according to different machines: 30 s for StepOne Plus, 31 s for 7300 and 34 s for 7500. If not otherwise specified, default to 30 s.
Data Analysis
1. Plot the standard curve based on Ct values and sample input quantity. The correlation coefficient (R²) of the standard curveshould be >0.98, with a slope between -3 and -3.5. The PCR amplification efficiency (E) generally falls between 90-120%.
2. The standard deviation (STD) of Ct values between replicate wells should be <0.2. The STD of Ct values for the sameexperiment across different batches should be <0.5 (when comparing the same experiment across batches, ensure the threshold setting is essentially consistent).
3. The melting curve of the amplification product shows no significant non-specific amplification products (non-specific peaks) or primer-dimer peaks (confirm by agarose gel electrophoresis if necessary). Furthermore, the Tm value of the meltingcurve is typically between 80-95°C.
4. Validation of Valid Ct: A valid amplification Ct value must be less than the Ct value of the no-template control curve, whileits melting curve must show no non-specific peaks.