Probes

Low signal values are mostly due to high background fluorescence (for example, check the baseline-corrected or ROX-corrected raw curves). In probe-based detection, this is often caused by:        

* Poor probe design:Leading to insufficient quenching by the quencher group.

* Improper pairing: Incorrect matching of the reporter and quencher groups.

* Low labeling efficiency: The probe's fluorescence labeling efficiency is too low.

Erratic peaks often result from contamination in the reaction system.   

* Confirm contamination using No Template Control (NTC) and No Reverse Transcriptase Control (NRC) results.

* Check potential sources such as water, oligonucleotides, enzymes, and the experiment settings.   

* Reagents exposed to strong light or high temperatures may degrade. It is recommended to compare results with fresh reagents.

Primers that have been resuspended may degrade or produce inconsistent results over time due to several common factors:

(1) Primer degradation: Resuspension in nuclease-contaminated water or solutions with non-neutral pH can lead to gradual chemical degradation or enzymatic cleavage, reducing primer integrity.

(2) Improper handling: Incomplete thawing, insufficient mixing, or repeated freeze-thaw cycles may result in uneven primer concentration, causing variability in pipetting and assay performance.

(3) Changes in other reaction components: Apparent primer failure may also be caused by deterioration or variation in other PCR reagents-most notably the DNA template - rather than the primer itself.

Recommended Best Practices:

(1) Resuspension buffer: Dissolve primers in nuclease-free 10 mM Tris-HCl (pH 7.5 - 8.0) to maximize stability.

(2) Aliquot and storage: Aliquot primer solutions into single-use or low-use volumes and store at -20°C to minimize freeze–thaw cycles.

(3) Thorough mixing before use: After thawing, vortex briefly and centrifuge to ensure a homogeneous solution prior to pipetting.

Generally not. Modern PCR techniques and high-temperature polymerases have been developed to solve most issues of amplification failure and low n efficiency. Failures are more commonly due to two main situations:

(1) RT-PCR: The quality of the RNA used in the RT reaction and the content of the target gene in the tissue/cell are critical.

For example, some repeat fragments, fragments with high GC content amplification, must use special amplification means to amplify.

(2) Amplification from the genome: Genes are single copies in the genome, so the amount of genomic DNA template must be strictly controlled. Too much genomic DNA can affect the Mg2+ concentration and pH in the reaction system.

No. Sequencing of non-specific fragments usually finds at least one primer sequence at both ends. Therefore non-specific amplification is typically the result of template contamination (e.g. contaminated genome in RNA) or unsuitable amplification conditions.