Probes

First check the baseline-corrected or ROX-corrected raw curves. Low signal values in the amplification curves are mostly due to high background fluorescence. In probe-based detection, high background fluorescence is often caused by poor probe design, leading to insufficient quenching by the quencher group, improper pairing of the reporter and quencher groups, or low fluorescence labeling efficiency of the probe.

Erratic peaks in melting curves can result from contamination in the reaction system. Confirm contamination using NTC and NRC results, and check potential sources such as water, oligos, enzymes, and the experiment settings. Reagents exposed to strong light or high temperatures may degrade, so it is recommended to compare results with fresh reagents.

If the water in which you dissolve the primers has a low pH or is contaminated with bacteria or nucleases, the primers will degrade. Inadequate thawing and mixing during use, and uneven liquid may also cause inaccurate amounts of primers. It is recommended to aliquot primers, avoid repeated freezing and thawing, and use 10 mM Tris pH 7.5 buffer to dissolve primers. There is another possibility that there is no problem with the primers, but that the quality of the materials used in PCR, especially the template, is not completely consistent with that used previously.

Basically not. Nowadays, various PCR amplification techniques and high-temperature polymerases have been developed to solve the problems of amplification failure and low amplification efficiency encountered in PCR amplification. For example, slot PCR amplifies gene fragments with very low copy number. Some repeat fragments, fragments with high GC content amplification, must use special amplification means to amplify.

Amplification does not come out, mainly the following two situations are more common.

(1) RT-PCR. many genes are difficult to amplify by conventional RT-PCR methods. the key to successful RT-PCR lies in the quality of the RNA of the RT reaction and the content of the target gene in the particular tissue and cell.

(2) Amplification from the genome. Generally, genes are single copies in the genome, and the genome needs to be used as a template in strictly controlled amounts. Too much genomic DNA can affect the Mg²⁺ concentration and pH in the reaction system.

No. We have analyzed some non-specific bands and sequencing found that at least one primer sequence can be found at both ends of these non-specific fragments. Therefore non-specific amplification is usually the result of template contamination (e.g. contaminated genome in RNA) or unsuitable amplification conditions.