Blue Universal SYBR Green Fast qPCR Mix with UDG HYA414
This product is a blue-colored universal SYBR Green reagent, convenient for sampling and using specially designed reference dye (ROX) for higher sensitivity and resolution. It is compatible with all models of fluorescence quantitative PCR instruments (including High ROX, Low ROX, and No ROX required instruments). The reagent introduces a dUTP/UDG system preventing the impact of amplification product contamination on qPCR and ensuring the accuracy of the results. UDG can quickly degrade pollutants containing U at room temperature, and UDG quickly deactivates when pre denatured at 95 ℃, without affecting the efficiency and sensitivity of qPCR. Hot start Taq DNA polymerase is used for amplification, which enhances the specificity of the product. Overall, this product provides a reliable and versatile solution for SYBR Green-based qPCR experiments.
Components
|
Components |
1 mL |
5 mL |
|
Blue Universal SYBR Green Fast qPCR Mix with UDG |
1mL |
5 × 1mL |
Instruments
No additional reference dye is required. Blue Universal SYBR Green Fast qPCR Mix with UDG is suited for all 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. Blue Universal SYBR Green Fast qPCR Mix with UDG, please thaw it before use, avoid direct sunlight, and store it in a dark place.
2. Blue Universal SYBR Green Fast qPCR Mix with UDG contains glycerol, please gently mix to avoid bubbles; Mix well and centrifuge before use. After use, immediately return it to the-20℃ refrigerator for storage.
3. This product contains DNA polymerase, so please put it on ice when using it. It can be temporarily stored at 4℃ after multiple uses in a short time. To maintain the quality of the product, it is recommended to avoid repeated freezing and thawing.
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 |
|
Blue Universal SYBR Green Fast qPCR Mix with UDG |
10uL |
|
Forward Primer (10 µM)* |
0.4 uL |
|
Reverse Primer(10 μM)* |
0.4 uL |
|
DNA template** |
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 |
|
UDG Reaction |
37 ℃ |
2 min |
1 |
|
Pre Denaturation |
95 ℃ |
3 min |
1 |
|
Denaturation |
95 ℃ |
5 s |
40 |
|
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.