PCR Simulator
Equations in this simulation
| N₀ | Starting copies of the template (as a power of 10) | double-stranded copies at the start | |
|---|---|---|---|
| E | Efficiency E (share of templates copied each cycle), % | 90 to 100% in a good reaction; lowered here when T_a is too close to the primers' Tm, and as the reaction runs out of primers and nucleotides | |
| n | cycle number | each cycle: denature at 95 °C, anneal at T_a, extend at 72 °C | |
| N_n | double-stranded copies after n cycles | levels off near 10¹² copies: the plateau |
With the current values:
| target copies | products of exactly the amplicon length | the first appear in cycle 3; until then the new strands run past the primer sites (long products), and their number only grows by 2 per cycle, so the target soon outnumbers them | |
|---|---|---|---|
| long products | copies with a strand longer than the amplicon | made from strands that run past a primer site; the counts below the picture follow every kind of molecule exactly |
With the current values:
| Tm | melting temperature of each primer | the basic formula for primers of 14 bases or more; the Primer Tm Calculator has the more accurate nearest-neighbor value | |
|---|---|---|---|
| T_a | Annealing temperature T_a (°C) | usually 3 to 5 °C below the lower Tm; much higher and the primers do not stay bound, much lower and they bind to the wrong places | |
| f_b | share of primers that anneal | 1 ÷ (1 + e^((T_a − Tm) ÷ 1.5)), a simple melting curve for the primer |
With the current values:
| L | amplicon length | in base pairs, primers included |
|---|
With the current values:
How to use the PCR simulator
- Paste a template and two primers, both written 5′ to 3′: the forward primer as it appears in the template, the reverse primer as it appears on the other strand. The simulator finds where they bind, the amplicon length and each primer's Tm; check a primer pair in more detail with the Primer Tm Calculator.
- Watch each cycle in the close-up: at 95 °C the two strands come apart, at the annealing temperature the primers attach at their sites, and at 72 °C the polymerase extends a new strand from each one. Below, the tube fills with copies: gray original templates, blue long products that run past the primer sites, green target amplicons of exactly the right length.
- Set the annealing temperature, efficiency and number of starting copies, and read the exact count of each kind of product after every cycle. The chart plots the copies on a log scale, where amplification is a straight line that bends into the plateau. To work out how many copies a given mass of DNA is, use the DNA Copy Number Calculator and its guide.
Frequently asked questions
How many copies does PCR make?
Each cycle can at most double the DNA, so n cycles give up to 2ⁿ copies per template: about a billion after 30 cycles. Real reactions run at 90 to 100% efficiency, N = N₀ (1 + E)ⁿ, and level off at about 10¹² copies as primers and nucleotides run out.
Why are the first products longer than the target?
In the first cycle each primer copies the original strand from its site to the end of the template, because nothing tells the polymerase to stop. Only when a primer binds one of those new strands, which ends at the other primer's site, is a strand of exactly the target length made. The first double-stranded target molecules appear in cycle 3, and after 30 cycles they make up all but a tiny fraction of the products: 2ⁿ − 2n per template, against 2n long products.
How do I choose the annealing temperature?
Start 3 to 5 °C below the lower Tm of the two primers. Too hot and the primers do not stay bound, so the efficiency collapses, which the simulator shows; too cold and they bind at partly matching places too, giving extra bands on a gel. A gradient PCR across several temperatures finds the best one.
What is Ct in qPCR?
In quantitative PCR a fluorescent dye reports the DNA as it is made, and Ct is the cycle at which the signal crosses a set threshold, like the dashed line on the chart. Each tenfold more starting template crosses about 3.3 cycles earlier, which is how qPCR measures how much was there. The ΔΔCt Calculator turns Ct values into fold changes.
It says WebGL is turned off.
The 3D view needs WebGL, which every current browser has. It can be switched off by hardware acceleration being disabled in the browser settings, or by a very old graphics driver. Turn hardware acceleration on, or try another browser.
Is anything uploaded?
No. The simulation is drawn by your own browser with WebGL; nothing is sent anywhere, and it keeps working offline once the page has loaded.