PCR product size calculator

What is a PCR product?

A PCR product, or amplicon, is the stretch of DNA that two primers copy between them. The forward primer anneals to one strand and the reverse primer to the other, and every cycle copies the template from one primer to the other. The product therefore starts with the forward primer sequence and ends with the reverse complement of the reverse primer, and both primers are part of its length.

How PCR product size is calculated

The calculator searches the template for the forward primer and for the reverse complement of the reverse primer. For every forward site that lies upstream of a reverse site it reports one product: its length in base pairs, its position on the template (1-based, inclusive), the position of each primer, the GC content of the amplicon, and the full amplicon sequence, ready to paste into a cloning map or to check against a gel.

Which way do I enter the reverse primer?

Enter both primers 5′→3′ exactly as ordered. A reverse primer is designed on the bottom strand, so its sequence as written does not appear in the top-strand template. The tool reverse-complements it for you before searching, so nothing needs to be flipped by hand. If a primer was accidentally designed on the wrong strand, try it in the other box: an amplicon then appears.

Degenerate primers and IUPAC codes

Ambiguity codes in a primer are expanded during matching, so R matches A or G, Nmatches any base, and so on. Degenerate primers for gene families or consensus targets can therefore bind several sites, and each combination of forward and reverse site is listed as a separate product. Ambiguity codes in the template itself are matched only literally.

Multiple products and mispriming

If a primer has more than one exact site, or the template contains repeats, several amplicons are listed, sorted from shortest to longest. This is a quick in silico check for extra bands on a gel: a second product close to the expected size will be hard to separate, and very short products usually indicate primer sites that overlap or face each other.

Why is no product found?

• A primer does not match. Matching is exact; one mismatched or missing base is enough. Check for typos and for a 5′ tail such as a restriction site that is not in the template.
• Wrong strand. The template may be the bottom strand of the region, or the primers may be swapped. Reverse-complement the template with thereverse complement tool or swap the primers.
• Primers face away from each other. The reverse site lies upstream of the forward site, so on a linear template nothing is amplified. On a circular plasmid this pair would amplify around the backbone instead.

Frequently asked questions

How is PCR product size calculated?

The amplicon runs from the first base of the forward primer binding site to the last base of the reverse primer binding site on the template, so its length is reverse-site end minus forward-site start plus one. Both primers are included in the product.

Do I enter the reverse primer as written on the tube?

Yes. Enter both primers 5' to 3' as they were ordered. The calculator reverse-complements the reverse primer itself and searches the template for that sequence.

Why does the calculator find no product?

Either a primer does not match the template exactly, or the reverse primer site lies upstream of the forward site. Check the template strand, that the reverse primer was designed on the opposite strand, and that there are no typos. Mismatches, including a single wrong base, are not tolerated.

Does it support degenerate primers?

Yes. IUPAC ambiguity codes in the primers (R, Y, S, W, K, M, B, D, H, V, N) match any base they stand for, so a degenerate primer can bind several sites and produce several products.

Can I paste a FASTA file or a GenBank sequence as the template?

Yes. A FASTA header line is ignored, and spaces, line breaks and position numbers are stripped before matching.