PCR Product Size Calculator

Calculate PCR product size, primer Tm, and expected band position on gel.

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What Sets Product Size and Yield

The amplicon spans from the 5′ end of the forward primer to the 5′ end of the reverse primer — primers are included in the product, which is a frequent source of off-by-a-few errors when designing to a target size.

Product = (reverse 5′ position) − (forward 5′ position) + 1

Amplification is exponential in principle, giving 2n copies after n cycles. In practice efficiency is below 100% and the reaction plateaus as reagents deplete and product reanneals faster than primers can bind.

CyclesTheoretical (2n)At 90% efficiency (1.9n)
101,024×613×
201.05 × 1063.75 × 105
301.07 × 1092.3 × 108
401.10 × 1012Plateau reached long before

The gap between the two columns widens dramatically with cycle number, which is why qPCR efficiency must be validated rather than assumed. A 10% efficiency shortfall costs nearly a fivefold difference by cycle 30.

Extension Time and Polymerase Choice

PolymeraseSpeedProofreadingUse for
Taq~1 kb/minNoRoutine, short products
Pfu~0.5 kb/minYesCloning, sequencing templates
Q5 / Phusion~1 kb/15–30 sYesLong or GC-rich targets

Extension time should be set from the product length and the polymerase's rate — roughly one minute per kilobase for Taq. Too short truncates long products; excessively long extension increases non-specific amplification and wastes cycle time.

Proofreading matters for anything that will be cloned or sequenced. Taq's error rate of roughly 1 in 104 bases accumulates across cycles, whereas high-fidelity enzymes are 50 to 100 times more accurate.

Worked Examples

Example 1: Gene fragment: pos 150-650
size=650-150+1=501bp
Result: Annealing ~53°C for 50% GC 20-mer primers
Standard diagnostic PCR product
Example 2: Short amplicon qPCR: 80-160bp
Ideal qPCR amplicon size
Result: Smaller = more efficient amplification
qPCR target: 80-150bp optimal
Example 3: Why efficiency matters in qPCR
30 cycles at 100% versus 90% efficiency
Result: 1.07 × 109 versus 2.3 × 108
Nearly fivefold difference. Standard curves exist precisely to measure efficiency rather than assume it.
Example 4: Extension time for a long amplicon
3.5 kb product with Taq at 1 kb/min
Result: About 3.5 minutes
Setting 1 minute would truncate most products. Long-range PCR often uses enzyme blends with extended processivity.

Common Mistakes

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Forgetting that primers are part of the product

The amplicon includes both primer sequences. Designing to a target size without counting them gives a product several tens of bases short.

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Assuming 100% amplification efficiency

Real efficiency is typically 90–100%. By cycle 30 a 10% shortfall means nearly fivefold less product, which matters enormously in quantitative work.

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Setting extension time too short for long products

Taq extends at roughly 1 kb per minute. A 3 kb product needs about three minutes, or the polymerase falls off before completing the strand.

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Using Taq for cloning or sequencing templates

Taq lacks proofreading and introduces roughly one error per 104 bases. Use a high-fidelity polymerase whenever the sequence matters.

Frequently Asked Questions

Wallace rule accuracy?
Tm = 4(G+C) + 2(A+T) °C. Accurate for short primers (<20nt) at 0.05M salt. Overestimates Tm for longer primers. Nearest-neighbor method (SantaLucia 1998) is more accurate for any length.
Primer design rules?
Length: 18-25 nt. Tm: 55-65°C. GC: 40-60%. Avoid: 3' GC clamp (2 G/C), hairpins (ΔG < -3kcal/mol), 3' self-complementarity. Tm difference between pair: < 5°C. Check for non-specific binding with BLAST.
Are primers included in the product size?
Yes. The amplicon runs from the 5′ end of the forward primer to the 5′ end of the reverse primer, so both primer sequences form part of it.
Why doesn't PCR yield 2n copies?
Efficiency is below 100%, and the reaction plateaus as primers and nucleotides deplete and product strands reanneal faster than primers can bind.
How long should extension be?
Roughly one minute per kilobase for Taq, less for fast high-fidelity enzymes. Too short truncates long products.
When do I need a proofreading polymerase?
Whenever the product will be cloned, sequenced or expressed. Taq introduces about one error per 104 bases, which accumulates across cycles.