Primer design and usage guidelines

Performing PCR primer design requires careful consideration and precise methodology to ensure the success of the PCR process. Optimal primer design is critical for achieving maximal specificity and efficiency, making it a cornerstone of accurate molecular biology experiments. "PCR primer design" involves selecting sequences that not only hybridize perfectly with the target DNA but also resist forming secondary structures or dimers that can hinder the PCR reaction.

Optimal primer sequences and appropriate primer concentrations are essential for maximal specificity and efficiency in PCR. The table, Primer design and usage guidelines, provides an overview of primer design and use for standard and multiplex PCR, as well as one-step RT-PCR. Molar conversions can be found in the table Molar conversions for PCR primers.

Primer design and usage guidelines
  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 information The Tm of all
primer pairs
should be similar
The Tm of all primer
pairs should be similar.
For optimal results,
the Tm should be
The Tm of all primer
pairs should be similar.
The Tshould not be
lower than the temperature
of the reverse transcription
(e.g., 50°C)
5°C below the
calculated Tm
5–8°C below the
calculated Tm (when
greater than 68°C)
or 3–6°C below the
calculated T 
(when 60–67°C)
5°C below the
calculated Tm
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
hybridizes 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.
A260 unit
20–30 µg 20–30 µg 20–30 µg

Molar conversions for PCR primers
Primer length pmol/µg 20 pmol
18mer 168 119 ng
20mer 152 132 ng
25mer 121 165 ng
30mer 101 198 ng

The following points should be considered when designing PCR primers and are common to all types of PCR:

  • Tm calculation: 2°C x (A+T) + 4°C x (G+C)
  • Avoid complementarity 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 3 or more Cs or Gs at the 3' end of the primer
  • Avoid complementarity within primers and between the primer pair
  • Avoid a T as ultimate base at the 3' end
  • Ensure primer sequence is unique for the template sequence
  • Use a concentration of 0.1–1.0 µM of each primer. For many applications, a primer concentration of 0.2 µM will be sufficient

Lyophilized primers should be dissolved in a small volume of distilled water or TE to make a concentrated stock solution. Prepare small aliquots of working solutions containing 10 pmol/µl to avoid repeated thawing and freezing. Store all primer solutions at –20°C. Primer quality can be checked on a denaturing polyacrylamide gel; a single band should be seen.