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Primer Pair Analyzer

Check a forward and reverse primer pair: each primer's Tm and GC, the difference between them, cross-dimer risk, and the product size if you paste the template.

Formula

ΔTm=∣Tmfwd−Tmrev∣\Delta T_{\mathrm{m}} = \left|T_{\mathrm{m}}^{\text{fwd}} - T_{\mathrm{m}}^{\text{rev}}\right|
L=xrev, end−xfwd, start+1L = x_{\text{rev, end}} - x_{\text{fwd, start}} + 1
TmT_{\mathrm{m}}
nearest-neighbour melting temperature of each primer
LL
length of the amplicon between the outer ends of the two primers, in bp

How it works

The primers of a PCR pair anneal at the same temperature, so their melting temperatures should be close. A commonly used guideline is to keep them within about 5 °C. Each is also checked against the usual design ranges, and the two are checked against each other for a dimer.

If you paste the template, the forward primer is searched for on the given strand and the reverse primer's reverse complement is searched for as well. When both are found facing each other, the amplicon length is reported. Only exact matches are found, so this does not predict priming at sites with mismatches.

Worked example

M13 forward primer GTAAAACGACGGCCAGT and reverse primer GGGTTTTCCCAGTCACGAC on a short illustrative cloning-site sequence.

  1. Nearest-neighbour Tm: 52.8 °C and 56.0 °C, a difference of 3.2 °C.
  2. The forward primer starts at base 7; the reverse primer's site ends at base 122.
  3. Size = 122 − 7 + 1 = 116 bp.

A 116 bp product with a 3.2 °C Tm difference, inside the usual 5 °C guideline.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Both primers are perfect matches to the template.
  • The Tm values use the concentration and salt entered; magnesium, dNTPs and additives shift them in a real reaction.
  • The reverse primer is entered 5′ → 3′, as ordered.

Common mistakes

  • Entering the reverse primer as its reverse complement, which finds no site.
  • Choosing a pair with balanced Tm but mismatched GC content at the 3′ ends.
  • Assuming a single product from a single exact match. A template with repeats can give several.

Related tools

Oligo and Primer AnalyzerLength, GC, Tm, weight and a design-guideline check for a primerOpen calculatorPrimer Tm Calculator (Four Methods)Primer melting temperature by four accepted methods, side by sideOpen calculatorPCR Product Size CalculatorAmplicon length from primer positions, with 5′ tailsOpen calculatorPCR Annealing Temperature CalculatorOptimal annealing temperature from primer Tm and productOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.