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HotStarTaq PCR Handbook

Table 7. General guidelines for standard PCR primers

Length: 1830 nucleotides G/C content: 4060% Tm: Simplified formula for estimating melting temperature (Tm): Tm = 2C x (A+T) + 4C x (G+C) Whenever possible, design primer pairs with similar Tm values. Optimal annealing temperatures may be above or below the estimated Tm. As a starting point, use an annealing temperature 5C below Tm. Sequence: Avoid complementarity of two or three bases at the 3' ends of primer pairs to reduce primerdimer formation.
Avoid mismatches between the 3' end of the primer and the target-template sequence.
Avoid runs of 3 or more G or C at the 3' end.
Avoid a 3'-end T. Primers with a T at the 3' end have a greater tolerance of mismatch.
Avoid complementary sequences within a primer sequence and between the primer pair.
Commercially available computer software (e.g., Primer Designer 1.0, Scientific Software, 1990; Oligo, Rychlik and Rhoads, 1989) can be used for primer design. Concentration: Spectrophotometric conversion for primers: 1 A260 unit = 2030 g/ml
Molar conversions:

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