1. Formulation Optimization: Composed of a genetically engineered hot-start enzyme and an optimized reaction buffer, specifically tailored for methylation detection applications.

2. Tolerance and compatibility: Exhibits strong sulfite tolerance, is compatible with templates spanning 25–70% GC content, and demonstrates robust uracil tolerance as well as the ability to recognize 3′-terminal mismatched bases.

3. Excellent performance: Multi‑target sensitivity and specificity, as well as the ability to detect low‑copy‑number targets, have been significantly enhanced; amplification curves exhibit outstanding linearity, and quantitative accuracy for target genes is high.

4. Convenient operation: A 2× pre-mix system requires only the addition of primers, probes, and template during use, significantly reducing pipetting steps and minimizing experimental errors.

1. Item number and specifications:
• JSR-E-206: 100 reactions specification, including 1 mL of 2× pre-mix;
• JSR-E-207: 500 reactions, including 5 × 1 mL of 2× pre-mix, for convenient aliquoting and use;

2. Core components:
• 2×Methy-qPCR Mix: Pre-mixed with hot-start DNA polymerase, dNTPs, an optimized buffer system, and more;
• 50×ROX High: High-concentration reference dye;
• 50×ROX Low: Low-concentration reference dye.

1. Storage conditions: Store frozen at -25°C to -15°C, protected from light; a one-year quality guarantee is provided from the date of sale.

2. Pre-use preparation: After removal, place on ice to thaw; once completely thawed, gently invert to mix, then briefly centrifuge before use.

3. Instrument‑use requirements: Prepare the reaction mixture, securely cap the tubes, and briefly centrifuge them to remove air bubbles that could interfere with fluorescence signal acquisition.

4. Post-use handling: Immediately replace the cap and store at −20°C to prevent prolonged exposure of the reagent to room temperature.

1. Standard 20 μL reaction system:
• 2×Methy-qPCR Mix: 10 μL
• Forward Primer (10 μM): 0.4 μL
• Reverse Primer (10 μM): 0.4 μL
• Probe (10 μM): 0.4 μL
• Template DNA: Add as needed (recommended 2 μL)
• 50×ROX (Low/High): 0.4 μL
• ddH₂O: bring to 20 μL

2. System Adjustment: The reaction volume can be scaled proportionally within the range of 5–25 μL; volumes exceeding 50 μL are not recommended.

3. Concentration Optimization: The final concentrations of primers and probes can be adjusted within the range of 0.1–1 μM to achieve optimal amplification performance.

1. ROX High : Compatible with Applied Biosystems® models 5700, 7000, 7300, 7700, 7900HT, StepOne™, StepOnePlus™, and others;

2. ROX Low : Compatible with Applied Biosystems® 7500, ViiA™ 7, QuantStudio™ 12K Flex, Agilent Mx3000P™, Mx3005P™, Mx4000™, and other models;

3. No ROX required : Compatible with Rotor-Gene™, Opticon™, Chromo4™, LightCycler® series, Bio-Rad CFX96, Illumina Eco™, and other models.

1. Applicable templates: DNA samples after sulfite conversion, methylated standards, plasmid templates, genomic DNA, etc.;

2. GC compatibility range: Compatible with templates having 25% to 70% GC content; targets with varying GC contents can all be stably amplified.

3. Tolerance characteristics: Exhibits excellent uracil tolerance, strong recognition of 3′-terminal mismatched bases, and is well suited to the base‑specific properties of DNA following bisulfite conversion.

4. Template recommendation: For methylation analysis, we recommend using our company’s JSR530 kit for bisulfite conversion.

1. Standard qPCR protocol (two-step method):
• Pre-denaturation: 95°C for 30 seconds, 1 cycle;
• Amplification cycles: 95°C denaturation for 10 seconds, followed by 60°C annealing/extension for 30 seconds with fluorescence acquisition, for a total of 35–45 cycles.

2. Parameter Optimization:
• Denaturation: For high-GC templates, the denaturation time can be extended to 3–5 minutes to ensure complete template denaturation.
• Annealing temperature: Adjust according to the primer/probe Tm value, with a range of 50–65°C; for initial experiments, a preliminary run at 60°C is recommended.
• Number of cycles: Typically 35–40 cycles; for low-copy-number samples, the cycle number may be increased to 45.

Abnormal amplification curves are often caused by issues such as instrument calibration, template concentration, air bubbles, or baseline settings; these can be systematically investigated and resolved.

1. The curve is not smooth. The reasons are system uncalibration, template concentration too low, and incorrect ROX selection.
Solution: Recalibrate the instrument’s fluorescence system, increase the template concentration, and verify that ROX is compatible with the instrument.

2. Curve breakage/decline The reasons are excessively high template concentration and an inappropriate baseline selection range.
Solution: Reduce the template concentration, reset the baseline range, and avoid regions with fluorescence fluctuations.

3. A sudden drop in individual curves The reason is that bubbles in the reaction tube interfere with fluorescence detection.
Solution: Before loading the sample, briefly centrifuge the PCR tube to remove air bubbles from the bottom and walls.

1. No amplification curve Common causes and solutions:
• Insufficient cycle number: It is recommended to set ≥40 cycles;
• Signal acquisition not configured: Verify that fluorescence detection is enabled during the annealing‑extension step of the two‑step protocol.
• Primer degradation: Prимер integrity is assessed by PAGE electrophoresis; if degradation is detected, the primer is resynthesized.
• Template concentration too low/degradation: Increase the template loading amount, or prepare the template anew.

2. High Ct value Common causes and solutions:
• Low amplification efficiency: Optimize reaction conditions, try a three-step amplification protocol, or redesign the primers.
• Low template concentration: reduce template dilution and increase the amount loaded.
• Overly long PCR products: It is recommended to keep the amplicon length between 80 and 150 bp.
• Presence of PCR inhibitors: Increase the template dilution factor, or re-purify the template.

Three common types of experimental issues correspond to different optimization strategies:

1. A clear amplification was observed in the negative control. ：
Cause: Contamination of the reaction system and non-specific primer design.
Solution: Replace the reagents, water, and primers; perform all procedures within a clean bench to minimize aerosol contamination; and redesign the primers to enhance specificity.

2. The standard curve exhibits poor linearity. ：
Causes: pipetting errors, degradation of standards, and excessively high template concentrations.
Solution: Increase the template dilution factor and the sample volume, re‑prepare the standards, and optimize the gradient concentration range.

3. The experiment has poor reproducibility. ：
Reasons: inconsistent sample volumes and excessively low template concentrations.
Solution: Use a pipette with higher precision, reduce the template dilution factor, or increase the aliquot volume; pre‑mix common reagents and then aliquot.