PCR Calculator
PCR helper — annealing temperature, extension time, product mass, and amplification.
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What it does
The PCR calculator handles four routine calculations that come up when planning an amplification: the annealing temperature to use for a given primer Tm, the extension time for a given product length and polymerase, the mass of product expected from a number of template copies, and the theoretical amplification fold for a given cycle count and efficiency. Each mode takes one or two inputs and is meant for setting up a run or troubleshooting one, not for replacing empirical optimization.
How it works
The annealing mode applies the standard heuristic of running about 3 to 5 degrees below the lower primer Tm, and the calculator uses Tm minus 5 degrees Celsius as a conservative default. Extension time follows the polymerase copying rate: time = product length in kilobases x rate, with about 60 seconds per kilobase for Taq, 120 seconds per kilobase for Pfu and 30 seconds per kilobase for Q5 or Phusion. Product mass converts molecule counts to grams using the average mass of a double-stranded base pair, about 650 daltons, so mass = copies x base pairs x 650 / 6.022 x 10^23, expressed in nanograms. Amplification fold follows exponential growth, fold = (1 + E)^n for n cycles at efficiency E, which reduces to 2^n at perfect doubling.
Worked example
With primers whose lower Tm is 60 degrees Celsius, the recommended annealing temperature is 55 degrees. A 1500 base pair product amplified with Taq needs 1.5 x 60 = 90 seconds of extension, which is 1 minute 30 seconds. Thirty cycles at 90% efficiency give 1.9^30, roughly 2.3 x 10^8 fold, whereas perfect doubling would give 2^30, about 1.07 x 10^9 fold. Starting from 1 million copies of a 1000 base pair target, the template itself amounts to only about 0.0011 nanograms, which is why amplification is needed before the product is visible on a gel.
When to use it
When a reaction gives no product, recalculate the annealing temperature from the actual primer Tm and run a gradient a few degrees either side of it before changing anything else. When switching from Taq to a proofreading enzyme such as Pfu, or to a fast enzyme such as Q5, recalculate extension time so a long amplicon is not truncated or a short one over-extended. When quantifying, use the product mass estimate to decide how much reaction volume to load on a gel or how far to dilute before a downstream assay, and use amplification fold to see why pushing beyond about 35 cycles adds smear and artefacts rather than yield.
FAQ
- How do I calculate the annealing temperature from primer Tm?
- Take the lower Tm of the two primers and subtract 3 to 5 degrees Celsius; this calculator uses 5 degrees as a safe starting point. If the reaction produces non-specific bands, raise the annealing temperature toward or slightly above the Tm to increase stringency, and if there is no product at all, lower it, ideally by running a temperature gradient.
- How long should the PCR extension time be?
- Scale it to the product length using the polymerase rate, roughly 1 minute per kilobase for Taq, 2 minutes per kilobase for Pfu, and 30 seconds per kilobase for Q5 or Phusion. Very short products still need a minimum step of a few seconds for the enzyme to complete synthesis, and for long or difficult templates a modest extra margin is safer than cutting the time fine.
- What is PCR efficiency and why is it not 100%?
- Efficiency is the fraction of template molecules successfully copied each cycle, where 100% means the amount of product doubles. Real reactions typically run at 80 to 95% because primer annealing is incomplete, secondary structure blocks the polymerase, and as cycles accumulate primers, nucleotides and enzyme activity are depleted while product strands re-anneal to each other, which is what produces the plateau phase.
- How many PCR cycles should I run?
- Between 25 and 35 cycles suits most reactions, with 25 to 30 for abundant template and 35 for scarce template. Beyond that the reaction has usually plateaued, so extra cycles add little product while giving more time for non-specific amplification, primer dimers and polymerase errors to accumulate.