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Oligo and Primer Analyzer

Analyze a primer or oligo: length, GC, GC clamp, runs and repeats, molecular weight and Tm by your choice of method, checked against common design ranges.

Formula

GC %=G+CN×100\mathrm{GC}\,\% = \dfrac{G + C}{N}\times 100
GC clamp=number of G or C in the last 5 bases\text{GC clamp} = \text{number of G or C in the last 5 bases}
Tm by the method you choose (see the Primer Tm calculator)T_{\mathrm{m}}\ \text{by the method you choose (see the Primer Tm calculator)}
NN
primer length in nucleotides
GC clamp\text{GC clamp}
G or C bases near the 3′ end, which anchor it to the template

How it works

A primer has to anneal specifically at the reaction temperature and be extended from its 3′ end. Common design guides reduce this to measurable properties: a length of about 18 to 30 bases, 40 to 60% GC, a melting temperature in a window that matches its partner, one to three G or C among the last five bases, and no long single-base runs or dinucleotide repeats.

This page measures each property and marks whether it lies inside the usual range, and lists the melting temperature by all four methods so you can see how much the choice matters. The ranges are rules of thumb, and they shift with the polymerase and the application, so a value outside a range is a prompt to look, not an error.

Worked example

The M13 forward (−20) primer GTAAAACGACGGCCAGT, nearest-neighbour Tm at 500 nM and 50 mM salt.

  1. Length 17 nt, GC 9 / 17 = 52.9%, Tm 52.8 °C.
  2. The last five bases are CCAGT, which contain 3 G or C.
  3. The longest single-base run is four A.

Length and the A run are outside the usual ranges (17 nt is below 18; AAAA is a run of four); GC, clamp and Tm are inside.

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

Assumptions

  • A single-stranded DNA oligo of A, C, G and T only.
  • Molecular weight assumes a 5′ hydroxyl, as for a synthetic oligo.
  • The guideline ranges are commonly quoted, not universal.

Common mistakes

  • Designing both primers of a pair with different Tm methods, so that the difference shown is partly the methods.
  • Ignoring a long run of one base because the other measures look good. Runs cause mis-priming and slippage.
  • Judging a primer by these measures alone. Specificity depends on the whole template, which this tool does not see.

Related tools

Primer Tm Calculator (Four Methods)Primer melting temperature by four accepted methods, side by sideOpen calculatorPrimer Hairpin and Dimer CheckerSelf-complementary stems and primer-dimer runs by sequence matchingOpen calculatorPrimer Pair AnalyzerTm match, GC, dimers and product size for a forward-reverse pairOpen calculatorDNA and RNA Molecular Weight CalculatorExact molecular weight of an oligo or sequence, ss or dsOpen calculator

Related equipment

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