PCR Run Time Calculator — Amplicon Size × Extension Rate

Core formula

The total PCR program run time is the sum of three parts:

Total time (s) = initial denaturation + N_cycles × (denaturation + annealing + extension) + final extension

The extension time per cycle is determined jointly by amplicon size and polymerase rate:

t_extension = max(t_min_extension, amplicon_bp / (rate_kb_per_min × 1000) × 60)

max(...) ensures that even for very short amplicons, the per-cycle extension time never falls below the polymerase’s recommended minimum.

The calculated extension time is rounded up to the nearest second: thermocycler programs only accept whole-second inputs, and it is always better to extend one second too long than one second too short (insufficient extension reduces yield; one extra second costs nothing). So 1234 bp at 1 kb/min gives 75 s, not 74.04 s.

Extension rate data sources

Preset parameters for each polymerase are taken from manufacturer technical manuals; rate is defined as single-strand synthesis speed:

Rates are condition-dependent: for templates with GC content ≥65%, secondary structures, or low dNTP concentrations, actual rates may fall below the nominal value. Users who have measured their own template can enter the empirical rate under “Custom”.

Applicability limits

1. Ramp time not included: this tool assumes instantaneous temperature transitions; actual instruments have ramp delays, so real run times are typically 10–25% longer than the calculated result.
2. Heated-lid pre-warming not included: some instruments require a 2–3 min heated-lid warm-up before initial denaturation; this is not accounted for.
3. Short-amplicon extension time is constrained by the minimum: for amplicons ≤ 500 bp, extension time is usually set by the minimum extension value rather than the length formula, so differences between polymerases narrow significantly in this range.
4. No cycle optimization or yield estimation — this tool calculates time only.

Common mistakes

Using template size instead of amplicon size Extension time depends on the amplicon (the region between the two primers), not on the total template DNA length. If you are amplifying an 800 bp target from a 5 kb plasmid, extension time is calculated from 800 bp, not 5000 bp.

Applying Taq’s 1 min/kb rule to high-fidelity polymerases Setting Phusion extension time to 1 min/kb wastes 30 s per cycle — 30 cycles means 15 min of unnecessary waiting. Conversely, using Phusion’s time for Taq will reduce yield. Update extension time whenever you switch polymerases.

Rounding extension time to the nearest minute instead of reading the actual seconds For KAPA HiFi (15 s/kb), a 2 kb amplicon requires only 30 s. If you habitually set 2 min, you waste 45 min across 30 cycles. This tool outputs whole seconds that can be entered directly into the thermocycler.

Worked examples

Example 1: Taq, 2 kb amplicon, 30 cycles

Conditions: initial denaturation 300 s, denaturation 30 s, annealing 30 s, extension rate 1 kb/min, minimum extension 30 s, final extension 420 s.

t_extension = max(30, 2000 / (1 × 1000) × 60) = max(30, 120) = 120 s
Total time = 300 + 30 × (30 + 30 + 120) + 420
           = 300 + 5400 + 420
           = 6120 s = 102 min

Example 2: Phusion, 500 bp amplicon, 30 cycles

Conditions: initial denaturation 30 s, denaturation 10 s, annealing 30 s, extension rate 2 kb/min, minimum extension 15 s, final extension 120 s.

t_extension = max(15, 500 / (2 × 1000) × 60) = max(15, 15) = 15 s
Total time = 30 + 30 × (10 + 30 + 15) + 120
           = 30 + 1650 + 120
           = 1800 s = 30 min

Both examples can be verified by hand calculation.