StarLighter Probe qPCR Master Mix (High GC)

StarLighter Probe qPCR Mix (High GC) is a specialized premix for probe-based qPCR. It contains a premium hot-start Taq DNA Polymerase with enhanced template affinity, combined with a buffer optimized for amplification of GC-rich targets. This effectively suppresses nonspecific amplification, significantly increasing qPCR efficiency and sensitivity. The kit generates excellent amplification curves across a wide concentration range, enabling accurate quantitative and qualitative analysis of target genes and SNP detection. It offers excellent reproducibility, high reliability, and compatibility with a wide range of qPCR probes. The reference dye ROX is provided with the kit.

This product is a 2× premix that only requires the addition of primers, probes, and templates. It is easy to use and compatible with rapid protocols, shortening detection time.

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;

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 probes should be located within the primer amplification region to avoid Overlap with the primer or form secondary structure. The Tm value should be 5 to 10°C higher than that of the primer to ensure that the probe anneals before the primer.
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:

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 a new template.

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

Answer: ① Contamination of the reaction system: Repeat the experiment with new reagents, water, primers, etc.; operate in a clean bench to reduce aerosol contamination.

② Improper primer design: Redesign and synthesize 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.

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.