How to use it
- Pick the polymerase you are using. ⚠️ The temperatures differ by enzyme — even two Taq-family enzymes here extend at 68 °C and 65 °C.
- Enter the whole amplicon length in bp. Only the extension time is computed from it; every other row is the protocol's own value.
- Program the table into the thermocycler. The three tinted rows are the repeated block, and the × beside them is the cycle count.
- The annealing temperature comes back as a range. Follow the Tm calculator link in that cell and work it out from your primers.
💡 The link under the table is the manufacturer's protocol these values came from. If the reaction misbehaves, start there.
Formula and cautions
Only one number here is calculated: the extension time. Every other row is the manufacturer's printed value, carried across unchanged.
Extension time
The enzyme's seconds per kilobase, times the amplicon length. Rounded up, because a thermocycler takes whole seconds.
- Extension
- t = (s/kb) × (amplicon bp ÷ 1000)
⚠️ Why there is a table per enzyme
The five protocols here carry three different extension temperatures — 72 °C, 68 °C and 65 °C — and three different denaturation temperatures, 94, 95 and 98 °C. A single 'usually this' line would be wrong for three of the five, so nothing is generalised and the protocols are carried as printed.
⛔ Why no annealing temperature is suggested
All five protocols here say in their own text to use the manufacturer's Tm calculator. Annealing depends on the primer sequences, the salt and the buffer, so the enzyme alone cannot settle it. The range each protocol prints is shown instead, with a link from that cell to the primer Tm tool.
Cautions
- These are routine PCR conditions. High GC, strong secondary structure, very low template or very long amplicons all need further optimisation, and the protocol text says how — follow the link.
- Measure the amplicon to the outer ends of both primers. Measuring only the gap between them makes the extension time short.
- Enzymes whose protocols were not opened are absent. A value written here without reading its protocol would be indistinguishable on screen from a sourced one.
- Hot start versions can have different initial denaturation conditions. Check the protocol for the product you actually have.
Frequently asked questions
Often used together
Master Mix Calculator
How much of each component to pipette for a batch of reactions, with water filling the rest.
Primer Tm calculator
Melting temperature for primers and oligos by the nearest-neighbour model.
PCR Plate Designer & Master Mix Calculator
Lay out a 96-well plate and get the mix volumes that go with it, on one printable page.