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查看更多产品信息 Luminaris Color HRM qPCR Master Mix - FAQs (K1032BID)
8 个常见问题解答
Using standardized kits such as the Luminaris Color HRM Master Mix eliminates most variables, ensuring reliable results. However, HRM analysis might be further improved by repeating the post-PCR melt curve/dissociation step with different settings: include heteroduplex formation, extend temperature range or use smaller/larger temperature increments. In addition, it is recommended to try a different primer pair, evaluate amplification efficiency, Cq values, retest the purity of DNA, ensure there is only one PCR product amplified, no primer dimers and other PCR artifacts.
DNA melting behavior is affected by salts in the reaction mix, so it is important that the concentration of buffer, Mg2+, and other salts is as uniform as possible in all samples. Therefore, it is recommended to use the same genomic DNA purification procedure for all samples subjected to HRM analysis. This avoids introduction of variations due to differing compositions of elution buffers used in different extraction methods. Diffrences in template amount can also contribute to different melting behaviour. Therefore we recommend using comparable amounts of template genomic DNA for all samples differing by no more than two Cq values, with the Cq values below 30.
We recommend to use 50-250 bp long PCR amplicons. Typically, shorter amplicons can distinguish the genotypes for a SNP better, especially for Type III and Type IV SNPs. This is simply because a single base variation affects the melting behavior of a 100 bp amplicon stronger than of a 500 bp amplicon, for example. In longer fragments, the risk of covering multiple mutations is also increased.
Positive control reactions should contain template DNA with a known sequence. In SNP genotyping experiments, this could be a sample with a known genotype. Positive control(s) for all genotypes should be included where possible to serve as a reference in melting curve comparison and assigning genotypes for test samples. In mutation scanning experiments, a sample with a wild type sequence could serve as a positive control. The controls should preferably have the same DNA concentration as their corresponding test samples. Control DNA should also be eluted and/or diluted in the same buffer as the samples.
A 3-step cycling protocol is recommended for the analysis of complicated (especially Type IV SNP) targets, amplicons longer than 200 bp, and amplicons with a primer annealing temperature that is less than 60 degrees C.
Luminaris Color HRM Master Mix contains third-generation EvaGreen fluorescent dye. The excitation and emission maxima of EvaGreen dye are at 500 nm and 530 nm, respectively, in DNA-bound state, and 471 nm without DNA.
Luminaris qPCR master mixes contain Hot Start Taq DNA Polymerase. This is a Taq DNA polymerase, which has been chemically modified by the addition of heat-labile groups to amino acid residues blocking enzyme activity. The Hot Start Taq DNA Polymerase has ultra low residual activity until thermally activated. Therefore reactions can be assembled at room temperature with no risk for the extension of non-specifically annealed primers or primer dimers and providing higher specificity for DNA amplification. The enzyme is activated by 10 minutes incubation at 95 degrees C.
When using a hot start polymerase it is not critical to start reactions immediately after setup. The Hot Start Taq DNA Polymerase in Luminaris qPCR Master Mixes has ultra low residual activity until thermally activated. Therefore reactions can be assembled and stored at room temperature for up to 72 hours with no risk for the extension of non-specifically annealed primers or primer dimers.