Frequently Asked Questions

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.

Tsingke ensures sample uniformity by isolating monoclonal clones. Each clone is validated by Sanger sequencing, covering the entire gene and at least 50 bp of the vector backbone upstream and downstream, which ensures the gene is correct and properly placed in the vector. The plasmid then undergoes NGS to verify the accuracy of the entire plasmid and detect any contamination. Before shipment, a strict QC review is conducted.

Cancer research probes, basic research probes, hematologic probes, pathogenic probes, genetic disease probes, methylation probes.

1.Primer Length: Primers should be 18-24 bases long to minimize the likelihood of primer dimer formation.

2.Tm Values: The melting temperatures (Tm) of all primers in the multiplex reaction should be similar to ensure uniform annealing.

3.3'-Nucleotide Complementarity: Avoid complementarity at the 3' ends of primers to prevent the formation of primer dimers.

4.Individual Optimization: Optimize each pair of primers independently before pooling them. Once pooled, mix and optimize them sequentially.