Adapted from TaqMan® One-Step RT-PCR Master Mix Reagents Kit Instruction
Design of Probes
Keep the G-C content in the 20 to 80% range. Avoid runs of an identical nucleotide. This is especially true for guanine, where runs of four or more Gs should be avoided. Do not put Gs on the 5´ end. ｓｅｌｅｃｔ the strand that gives the probe more Cs than Gs.
For single-probe assays, Tm should be 68 to 70 °C when using Primer Express software.
Design of Primers
Choose the primers after the probe. Design the primers as close as possible to the probe without overlapping the probe. Keep the G-C content in the 20 to 80% range. Avoid runs of an identical nucleotide. This is especially true for guanine, where runs of four or more Gs should be avoided. When using Primer Express software, the Tm should be 58 to 60 °C. The five nucleotides at the 3´ end should have no more than two G and/or C bases.
Keep the size of PCR product or amplicon to 50 - 150 bp. The shorter amplicons work most efficiently: the most consistent results are obtained for amplicon size ranges from 50 to 150 bp.Both primers and probes can be designed using the Primer Express software provided by Applied Biosystems.
