501
bp
0.501
kb
461
bp
500
bp
251
bp
501
bp
0.501
kb
461
bp
500
bp
251
bp
The PCR Product Size Calculator determines the expected amplicon size based on the genomic positions of your forward and reverse primer binding sites. Knowing the expected product size is essential for validating PCR reactions on agarose gels, selecting the appropriate gel percentage, and choosing the correct DNA ladder for size estimation. Simply enter the 5' binding position of the forward primer and the 3' binding position of the reverse primer on the template sequence.
The PCR product size is calculated from the primer binding positions on the template:
Product Size (bp) = |End Position − Start Position| + 1
Where:
The +1 accounts for inclusive counting of both boundary nucleotides. The product includes both primer sequences and the intervening template region.
Inputs
Results
Primers binding at positions 150 and 850 produce a 701 bp amplicon, suitable for a 1% agarose gel.
Inputs
Results
A 221 bp amplicon is ideal for quick diagnostic PCR and can be resolved on a 2% agarose gel.
Choose gel percentage based on product size: 0.5–0.8% for fragments >5 kb; 1% for 0.5–5 kb (most common); 1.5% for 0.2–1 kb; 2–3% for fragments <300 bp. For very small products (<100 bp), use polyacrylamide gels or high-resolution agarose. MetaPhor agarose provides better resolution for small size differences.
Possible reasons: (1) Introns — if you designed primers from mRNA sequence but amplifying genomic DNA, introns add extra length; (2) non-specific amplification — primers binding elsewhere in the genome; (3) splice variants — alternative transcripts with different exon compositions; (4) insertions/deletions in the template sequence; (5) primer mispriming at an unintended site.
Use the NCBI Primer-BLAST tool to find your primer binding sites on the reference genome or sequence. The tool reports the exact start and end coordinates. Alternatively, perform a local alignment (BLAST) of your primer sequences against the target and note the matching positions. Most sequence visualization tools also have a primer search feature.
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