Product Features

1. Ultra-high plateau value
2. Perfect balance between sensitivity and specificity
3. Universality across all platforms

Key Data

Ultra-high plateau value

Hela cDNA was used as a template to amplify six different genes with Q712 and SYBR qPCR reagents from other brands (Supplier A, B, C, D).

Perfect balance between sensitivity and specificity

Figure A shows eight 10-fold gradient dilutions of the pUC19 plasmid, and Figure B displays the standard curve obtained based on the CT values in Figure A. In Figure C, Hela cell cDNA was used as a template, and six different genes were amplified under the same reaction conditions using Q712 and SYBR qPCR reagents from other brands (Supplier A, B, C, D).

Universality across all platforms

Q712 was used to amplify the EGFR gene on different qPCR devices (Type: ABI Stepone, ABI QuantStudio 3, and Roche LightCycler 480) with excellent quantitative results.

Components Components Q712-02 (500 rxns/20 ul reaction) Q712-03 (2,500 rxns/20 ul reaction) 2 × Taq Pro Universal SYBR qPCR Master Mix* 4 × 1.25 ml 5 × Q712-02

*Includes dNTP, Mg2 + , Taq Pro DNA Polymerase, SYBR Green I, and Specific ROX Reference Dye, etc.

Storage

Store at -30 ~ -15°C and protect from light for up to 18 months. The master mix can be stored stably at 2 ~ 8°C for 6 months and, after thawing, protected from light. Transport at ≤ 0°C.

FAQs

Q1: What should be considered when designing primers for qPCR?

A1: (1) The recommended size of amplification products is 80 – 200 bp. Smaller sizes result in higher efficiency, but the product size should be above 80 bp for differentiation from primer dimers.

(2) Avoid GC-rich or AT-rich regions at the 3′ end.

(3) The last base should be G or C instead of T.

(4) The temperature difference (Tm) between forward and reverse primers should not exceed 1°C.

(5) GC content should be 40–60%.

(6) For probe-based qPCR, the Tm of probes should be 8°C–10°C higher than that of primers.

Q2: Is dilution of the cDNA template necessary? What is the dilution factor for qPCR?

A2: There is no reference dilution factor. Generally, the Ct value increases by 3.3 for each 10-fold dilution of the cDNA. Plan dilutions accordingly using the stock cDNA solution, 10-fold diluted solution, and 100-fold diluted solution for qPCR. Ensure that the input amount does not exceed 1/10th of the qPCR system, as cDNA may contain components that inhibit qPCR in large volumes.

Q3: Abnormal shapes of amplification curves.

A3: (1) Uneven amplification curve: Signal is weak, resulting in an uneven curve after system calibration. Repeat the experiment after increasing the template concentration.

(2) Distorted or reduced amplification curve: The baseline of the last cycle is higher than the Ct value due to high template concentration. Reduce the number of cycles (Ct value – 4) and reanalyze the data.

(3) Sharp drops in specific amplification curves: Residual bubbles in the reaction tube burst with increasing temperature, causing a sudden decrease in fluorescence intensity detected by the device. Examine the reaction tubes carefully for remaining bubbles before testing.

(4) Rough and discontinuous amplification curve: ROX was not properly added. Calibrate the reference dye.

Q4: Amplification efficiency below 90% or above 120%, and a weak linear relationship in the standard curve.

A4: (1) Sample loading error. Increase the template dilution factor and sample volume. Use different dilution gradients for more accurate concentrations.

(2) The standard is degraded. Prepare a new standard and repeat the experiment.

(3) The template concentration is too high and can hinder the reaction. Increase the template dilution factor.

(4) Weak specificity of primer amplification. Redesign primers and repeat the experiment.

Q5: No amplification curve at the end of the reaction.

A5: (1) Insufficient reaction cycles: Generally, the number of cycles is set to 40. Too many cycles will increase background signals and decrease data reliability.

(2) Check if the signal acquisition step is set up in the program: Two-step qPCR usually sets the signal acquisition during annealing and extension stages; three-step qPCR sets the signal acquisition during the 72°C extension stage.

(3) Unsuitable primers: Redesign primers. Check if the primers are degraded: The integrity of long-stored primers should be tested by PAGE electrophoresis to eliminate potential degradation.

(4) Very low template concentrations: Reduce the dilution factor and repeat the experiment. For unknown concentration samples, first test the highest concentration.

(5) Template degradation: Prepare new templates and repeat the experiment.

Q6: Criteria for the validity of Ct values.

A6: (1) Single peak in the melt curve (dye-based).

(2) Standard deviation (STD) of Ct values in the exponential phase of the amplification curve between copy wells is < 0.2.

(3) Appropriate threshold.

(4) Experiment with no-template control (NTC) shows no or negligible aerosol contamination.

(5) Experiment without reverse transcription control (NRT) indicates no or negligible contamination by residual genomic DNAs.

(6) Amplification efficiency (e) meets the approximate criteria of 95–105% or 90–120%; the correlation coefficient (R2) of the standard curve is above 0.98.

Q7: Ct values are very high.

A7: (1) Amplification efficiency is very low. Optimize reaction conditions using a three-step qPCR program or redesign synthetic primers.

(2) Template concentration is very low. Reduce the dilution factor and repeat the experiment. For unknown concentration samples, first test the highest concentration.

(3) Template degradation. Prepare new templates and repeat the experiment.

(4) PCR product is very long. The recommended PCR product size is 80 bp–150 bp.

(5) The system contains PCR inhibitors. Inhibitors are usually introduced by the template. Increase the template dilution factor or prepare a new template and repeat the experiment.

Q8: If NTC has a Ct value, can the Ct value of the target gene still be used?

A8: NTC amplification usually involves two scenarios:

(1) Melt curve peak model does not overlap with the target gene. NTC typically has a lower Tm than the target gene. In this case, the Ct value of NTC is due to primer dimers and does not affect the Ct value of the target gene.

(2) Melt curve peak model overlaps with the target gene. NTC has the same Tm as the target gene. This indicates contamination by aerosols. To determine if the Ct value of the target gene can still be used, calculate the Ct value difference (ΔCt) between the target gene and NTC. If ΔCt is within the acceptable range, the target gene Ct value can still be used.

Q9: What should be done if the amplification curve is not smooth, or if the baseline is not stable?

A9: (1) Check if the reaction tube contains bubbles. Bubbles can affect the smoothness of the amplification curve and the stability of the baseline.

(2) Check if the template or primer concentration is too high. Adjust the template and primer concentrations within the recommended range.

(3) Inspect the temperature uniformity of the thermal cycler. Uneven temperatures can lead to uneven amplification curves.

(4) Verify the integrity of the reagents. Expired or degraded reagents may cause irregular amplification curves.

(5) Ensure proper mixing of the reaction components before loading into the PCR instrument.

Q10: Can SYBR Green I be replaced with another dye?

A10: SYBR Green I is a fluorescent dye that binds to double-stranded DNA. It can be replaced with other similar dyes, but the replacement dye should have similar properties, such as low interference with PCR reactions, high fluorescence intensity when bound to DNA, and a low affinity for single-stranded DNA. It’s essential to validate the performance of the replacement dye in your specific qPCR system before routine use.

Note: This information is provided as general guidance. Always refer to the specific protocols and guidelines provided by the manufacturer of the qPCR reagents and instruments used in your experiments.