StarLighter Taq Pro Probe Multiplex qPCR Master Mix (UDG)

Product Introduction

StarLighter U+ Taq Pro Probe Multiplex qPCR Mix is a universal reagent for probe-based multiplex real-time PCR (qPCR) reactions. The 2× StarLighter U+ Taq Pro Probe Multiplex qPCR Mix contains all components except template, primers, and probes. The optimized hot-start Taq Pro DNA Polymerase, combined with a buffer optimized for multiplex reactions, effectively inhibits nonspecific amplification, significantly increasing qPCR amplification efficiency and multiplex detection sensitivity. The reference dye ROX is provided with the kit.

Before use, this product requires only the addition of primers, template, probe, ROX (depending on the instrument model), and water. The kit produces excellent amplification curves across a wide concentration range, enabling accurate quantitative, qualitative, and SNP detection of multiple target genes. It offers excellent reproducibility, high reliability, and compatibility with a wide variety of qPCR probes. The reagent incorporates a dUTP/UDG anti-contamination system, which operates at room temperature, eliminating the effects of amplification product contamination on qPCR and ensuring accurate results.

Product components

Note: Please store at the recommended storage temperature and avoid repeated freezing and thawing.

Storage and transportation conditions

Store at -25 ~ -15℃, transport at < 0℃.

Product Application

Gene expression analysis;

Low copy gene detection;

SNP and allele typing;

validation of sequencing and microarray results;

Precautions

1. Always ensure the product is completely melted and mixed thoroughly before use.
2. Centrifuge before loading to avoid air bubbles in the PCR tube interfering with the fluorescent signal.
3. Use appropriate primer design software to design primers and Taqman probes. The Taqman probe should be located within the region amplified by the primers to avoid overlap or secondary structure formation. The Tm value should be 5–10°C higher than that of the primers to ensure that the probe anneals before the primers.
4. Confirm that the quencher (e.g., BHQ, TAMRA) and reporter (e.g., FAM, HEX) groups match the instrument detection channel.
5. Select the appropriate ROX according to different instruments.

Instrument and ROX Reference Dye Comparison Table:

experiment process

1. Reaction system preparation
   • Before preparation, ensure that all components are thoroughly mixed;
   • Prepare the reaction system according to the following table:

2. Reaction procedure

qPCR standard procedure

1. a. Set the initial denaturation time between 30 seconds and 5 minutes. If the template has a high GC content, the initial denaturation time can be extended to 5 minutes.
2. b. Denaturation time: 10 seconds for the standard program and 3 to 10 seconds for the fast program.
3. c. The annealing/extension temperature needs to be determined based on the Tm values of the primers and probes, generally 50 to 65°C. 60°C can be used for preliminary experiments for the first time.
4. d. Annealing/extension time can be adjusted between 20 and 45 seconds depending on the length of the amplified fragment.

Common Problems and Solutions

1. What are the causes and solutions for abnormal amplification curves?

Answer: ① If the amplification curve is not smooth, you need to recalibrate the system, increase the template concentration, and check whether ROX is used incorrectly.

②If the amplification curve breaks or declines, it may be caused by too high template concentration or inappropriate baseline selection range. You can reduce the template or reset the baseline.

③If the individual amplification curve drops suddenly, there may be bubbles in the reaction tube, so it should be centrifuged before loading.

2. What should I do if the reaction ends with no amplification curve?

Answer: ① It may be that the number of cycles is not enough: 40 cycles are generally set, but too many cycles will increase the background signal and reduce the credibility of the data.

② Confirm whether signal acquisition is set in the program: the two-step method generally collects signals during the annealing/extension stage; the three-step method collects signals during the extension stage.

③ Primer degradation: PAGE detection excludes the possibility of its degradation.

④The template concentration is too low: reduce the dilution and repeat the experiment, starting with a high concentration.

⑤ Template degradation: re-prepare the template.

3. Ct What if the value is too high?

Answer: ① Low amplification efficiency: Optimize reaction conditions, try three-step amplification or redesign synthetic primers.

② Low template concentration: Reduce the template dilution and start with high concentration.

③ Template degradation: re-prepare the template.

④The PCR product is too long: The recommended PCR product length is 80 to 150 bp.

⑤ PCR inhibitors are present: If the template is involved, increase the template dilution multiple or prepare the template again.

4. What should I do if the negative control shows obvious amplification?

Answer: ① The primer design is unreasonable: redesign and synthesize the primers.

5. What should I do if the standard curve has poor linearity?

Answer: ① Sample addition error: increase the template dilution multiple and increase the sample addition volume.

②Degradation of standard sample: re-prepare the standard sample.

③The template concentration is too high: increase the template dilution multiple.

6. What to do if the experiment has poor reproducibility?

Answer: ① Inconsistent sample volume: Use a better pipette; dilute the template by a high multiple and increase the sample volume appropriately.

②The template concentration is too low: The lower the template concentration, the worse the repeatability. Reduce the template dilution or increase the sample volume.