Maximizing PCR and RT-PCR Success - Primer design

14 PCR Brochure 10/2016 Primer design Optimal primer sequences and appropriate primer concentrations are essential for maximal specificity and efficiency in PCR. Table 2 provides an overview of primer design and use for standard and multiplex PCR, as well as one-step RT-PCR. Standard PCR Multiplex PCR One-step RT-PCR Length 18–30 nt 21–30 nt 18–30 nt GC content 40–60% 40–60% 40–60% Tm calculation 2°C x (A+T) + 4°C x (G+C) 2°C x (A+T) + 4°C x (G+C) 2°C x (A+T) + 4°C x (G+C) The Tm of all primer pairs The Tm of all primer pairs The Tm of all primer pairs should be similar. should be similar. should be similar. For optimal results, the Tm should The Tm should not be lower than be between 60 and 88°C. the temperature of the reverse transcription (e.g., 50°C). Estimating optimal 5°C below the calculated Tm 5–8°C below the calculated 5°C below the calculated Tm annealing temperature Tm (when greater than 68°C) 3–6°C below the calculated Tm (when 60–67°C) Location – – To prevent detection of gDNA: Primer hybridizes to the 3' end of one exon and the 5' end of the adjacent exon. Alternatively, the primer hybridized to a flanking region that contains at least one intron. If only the mRNA sequence is known, choose primer annealing sites that are 300–400 bp apart. Sequence Avoid complimentarity in the 2–3 bases at the 3' end of the primer pairs. Avoid mismatches between the 3' end of the primer and the template. Avoid runs of three or more C at the 3' end of the primer. Avoid complimentarity within primers and between the primer pair. Avoid a T at the 3' end. Ensure primer sequence is unique for your template sequence. Concentration, A260 20–30 µg 20–30 µg 20–30 µg unit equivalence Input 0.1–0.5 µM of each primer Input 0.2 µM of each primer Input 0.5–1 µM of each primer (0.2 µM recommended) (0.6 µM recommended) Storage Dissolved in TE, store at –20°C Dissolved in TE, store at –20°C Dissolved in TE, store at –20°C Table 2. Guidelines for the design and use of primers

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