Question 1

What math does this calculator use?

Accepted Answer

It uses the standard cycle-by-cycle recursion for PCR fidelity. Each cycle, every existing molecule is preserved and a new copy is made with probability (1 − e·L) of being error-free, where e is the polymerase per-base error rate and L is the amplicon length. The expected fraction of error-free molecules after n cycles is (1 − e·L/2)^n. This is the same model used by NEB’s PCR Fidelity Estimator and is the first-order approximation from Sun (1995).

Question 2

Where do the default error rates come from?

Accepted Answer

The presets are drawn from published manufacturer literature and primary references: Q5 and Phusion from NEB product sheets, KAPA HiFi from the Roche/KAPA datasheet, Platinum SuperFi II from Thermo Fisher, Pfu from Cline et al. (NAR 1996), and Taq from Barnes (PNAS 1994). If you have a lab-measured error rate, switch to the Custom option and enter it.

Question 3

Why is fidelity higher than I expected?

Accepted Answer

Because only half of the population is newly synthesized on any given cycle — the other half are parent molecules inherited from the previous cycle. This is why the effective error compounding per cycle is e·L/2, not e·L. For typical high-fidelity polymerases (e ~ 10⁻⁶) at 1 kb and 30 cycles, you should see ≥99% error-free product.

Question 4

Why does fidelity drop so fast for Taq?

Accepted Answer

Taq’s per-base error rate is ~3×10⁻⁵ — roughly 50–100× higher than a modern high-fidelity enzyme. For a 1 kb amplicon that is ~1.4% loss per cycle, and 30 cycles compounds it to 30–50% loss depending on the exact rate. For any cloning, NGS, or sequencing-grade work, use a proofreading enzyme (Q5, Phusion, KAPA HiFi, or Platinum SuperFi II).

Question 5

What does "cycles ≥ 95% fidelity" mean?

Accepted Answer

It is the largest number of cycles at which the expected fraction of error-free molecules is still above 95%. Running more cycles than this means more than 5% of your product will carry at least one substitution. This is a useful ceiling for cloning, single-molecule sequencing, or variant discovery.

Question 6

Does this account for recombination or amplification bias?

Accepted Answer

No. The model assumes every molecule duplicates in every cycle (ideal exponential amplification) and that errors are independent. It does not model chimera formation, GC-content amplification bias, or polymerase stalling. For long (≥10 kb) amplicons or GC-extreme templates, use the estimate as an upper bound on achievable fidelity, not a prediction.

Question 7

Does my data leave the browser?

Accepted Answer

No. All computation runs locally in JavaScript. Nothing is sent to any server. You can verify by disconnecting from the internet and reloading the tool.

Question 8

How should I cite this tool?

Accepted Answer

See the “How to cite” section on this page for a ready-to-paste methods statement and citation. The underlying math follows Sun (1995) and manufacturer-published error rates (Barnes 1994; Cline et al. 1996; NEB product documentation).

Polymerase	Error rate	Final % correct	Cycles ≥ 95%	Cycles ≥ 99%
KAPA HiFi	2.8e-7	99.4%	30	30
Platinum SuperFi II	3.0e-7	99.3%	30	30
Phusion High-Fidelity	4.4e-7	99.0%	30	30
Q5 High-Fidelity	5.3e-7	98.8%	30	25
Pfu	1.3e-6	97.1%	30	10
OneTaq Hot Start	2.0e-5	63.5%	3	0
Taq	2.9e-5	52.3%	2	0

PCR Fidelity Estimator

Fidelity Estimate

Expected molecules after 30 cycles

Fidelity across cycles

Comparison across preset polymerases (30 cycles, 1500 bp)

What is PCR fidelity?

The cycle-by-cycle recursion

Choosing a polymerase

Frequently Asked Questions

Next Steps

Thermal Cyclers

Gel Electrophoresis Systems

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