Frequently Asked Questions

In multiplex PCR, multiple primer pairs are used simultaneously, making optimization of reaction conditions critical for balanced and specific amplification.

(1) Annealing temperature: Each primer pair has its own optimal annealing temperature. For multiplex reactions, the annealing temperature is typically set based on the lowest melting temperature (Tm) among all primers to ensure efficient binding and amplification across targets.

(2) Cycle number: Using the minimum number of PCR cycles required to obtain sufficient product helps maintain amplification efficiency and specificity, while reducing nonspecific products and amplification bias between targets.

Careful optimization of both annealing temperature and cycle number is essential for reliable and reproducible multiplex PCR performance.

In multiplex PCR, multiple targets are amplified simultaneously, increasing competition for key reagents such as DNA polymerase, dNTPs, and primers. If reagent concentrations are not properly optimized, amplification efficiency and balance between targets may be compromised.

Additionally, the simultaneous synthesis of multiple amplicons increases the overall demand on the polymerase, often requiring longer extension times than single-target PCR to ensure complete and efficient synthesis of all products.

Optimizing reagent concentrations and extension time is therefore essential to achieve uniform amplification, high specificity, and reliable results in multiplex PCR assays.

For multiplex PCR, primers are typically designed to be 18-24 nucleotides in length. This range provides a balance between specificity and efficient annealing, while minimizing the risk of primer-dimer formation. Longer primers are generally more prone to secondary structure formation and non-specific interactions, which can negatively affect multiplex PCR performance.

This issue is most commonly associated with the inherent limitations of chemical oligonucleotide synthesis. Synthetic primers are not 100% pure, and trace amounts of truncated or failure sequences may be present. These minor impurities can occasionally participate in PCR amplification and be carried through cloning and sequencing.

We recommend the following actions:

(1) Sequence additional clones: Re-pick and sequence one or more additional colonies. In most cases, the initially sequenced clone originates from a PCR product amplified by a minor impurity, while other clones contain the correct sequence.

(2) Contact technical support if the issue persists: If the same primer-region error is observed consistently in 2-3 independent clones, the primer batch itself may be the source. Please contact our technical support team for further assistance. We will arrange prompt resynthesis or replacement of the primer at no additional cost.

We specialize in synthesizing RNA oligos up to 180 nucleotides in length. Types mainly include: siRNA, sgRNA, miRNA, ASO, and various modified RNAs.

All RNA oligonucleotides undergo rigorous quality control:

(1) Mass spectrometry (MS): Every batch is verified by MS to confirm identity and mass accuracy.

(2) HPLC purification: Available for applications requiring >90% purity.

(3) Nuclease-free and integrity checks: Gel electrophoresis and functional validation are optional upon request.

We guarantee sequence accuracy, modification integrity, and high batch-to-batch reproducibility.