| Amplicon Size. |
| PCR |
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| qPCR |
Ideally, the amplicon should be between 100 bp and 150 bp since longer products do not amplify as efficiently and we want to ensure that qPCR reaction efficiency is as close to 100% as possible.
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| Annealing Temperature. |
| PCR |
|
| qPCR |
Depending on the qPCR results the annealing temperature should be increased or decreased in 2-3°C increments to optimize further. This can be done in a single experiment using a thermal gradient or a range of annealing temperatures can be tested using multiple qPCR experiments.
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| Plate Color. Does it really matter? |
| PCR |
Plate color is irrelavent for PCR. |
| qPCR |
We recommend that opaque white qPCR plates are used for Real-Time PCR analysis. The white color elimates cross-talk and improves the efficiency of fluorescent detection thereby increasing assay sensitiviy and well-to-well consistency.
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| Primer Concentration. |
| PCR |
Start by using the primer concentration recommended by the manufacturer's specifications. If optimization is required, stay within the guidelines of the qARTA User Protocol and use 25 mM titrations to adjust primer concentration. |
| qPCR |
| Template Amount. |
| PCR |
When DNA is used as the starting template, nanogram amounts of cloned template, up to microgram amounts of genomic DNA, or up to 20,000 target copies can be a good starting point for optimization. However, even very low levels of sample (i.e., mRNA from tens of cells, DNA from single cells or individual viral genomes) may be sufficient for PCR amplification.
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| qPCR |
| Template Quality. |
| PCR |
It is essential that the nucleic acid is sufficiently pure for qPCR analysis. Template contamination (ie. genomic DNA, protein, carbohydrates or organic solvents) can have a huge impact on assay reliability and reproducibility. Template quality should be determined by spectrophotometry, microfluidics, or PAGE.
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| qPCR |
PCR and qPCR Optimatization Guide