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PCR Product Size Calculator

Calculate the length of a PCR product from the template positions of the two primers' 5′ bases, including any 5′ tails such as restriction sites added to the primers.

Formula

L=xrev−xfwd+1L = x_{\text{rev}} - x_{\text{fwd}} + 1
Ltails=L+ℓfwd tail+ℓrev tailL_{\text{tails}} = L + \ell_{\text{fwd tail}} + \ell_{\text{rev tail}}
xfwdx_{\text{fwd}}
template coordinate of the forward primer's 5′ base
xrevx_{\text{rev}}
template coordinate of the reverse primer's 5′ base, which is the larger number

How it works

The product of a PCR runs from the 5′ end of the forward primer to the 5′ end of the reverse primer, because each primer is incorporated at the end of its strand. If both positions are read as coordinates on the same strand of the template, the length is their difference plus one.

A 5′ tail that does not match the template, such as a restriction site or a Gibson overlap, is incorporated into the product, so it adds its length to the amplicon once the first cycles have copied it.

Worked example

The forward primer starts at template position 101 and the reverse primer's 5′ base is at position 420.

  1. Size = 420 − 101 + 1 = 320 bp.

A 320 bp product. With 6 and 8 nucleotide tails the final amplicon would be 334 bp.

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

Assumptions

  • Both positions are on the same coordinate system and strand.
  • The primers anneal at those positions with no insertion or deletion.

Common mistakes

  • Using a primer's 3′ end as its position, which is wrong by the primer length.
  • Forgetting the tails when you check a product by gel, so it runs larger than predicted.
  • Mixing coordinates of different reference sequences or versions.

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Related equipment

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