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100 rxn (20 μl/rxn)1. Aliquot reagents to avoid risk of contamination and repeated freeze-thaw cycles.2. Avoid high primer concentrations, a concentration of 100 nM to 300 nM of each primer usually gives the best results.3. Probe concentration may require optimization, typically a 3:1 to 1:1 primer:probe ratio will give good amplification.4. A very effective way to get tight Ct among replicates is to reduce pipetting error, this can be achieved by: 1) preparing amplicon specific pre-mix, 2) using repeating pipettes and 3) keeping the pipetting volume within the manufacturer's suggested range.5. For optimal results, it is recommended that the primers are 18-22 nucleotides in length with a Tm of 58°C-60°C and the size of target is about 100-250 bp.