Calculator Inputs
Formula Used
Base extension time = (Amplicon length in base pairs ÷ Polymerase speed in bp/min) × 60
Adjusted extension time = Base time × GC factor × Template quality factor × Assay complexity factor × Safety factor
Recommended per-cycle elongation = Adjusted extension time rounded up to your selected increment
Final extension time = Greater of (Per-cycle elongation × Final extension multiplier) or Minimum final extension
Total elongation-stage time = (Per-cycle elongation × Cycle count) + Final extension time
How to Use This Calculator
- Enter your amplicon size in bp or kb.
- Select a polymerase preset, or choose a custom extension speed.
- Add GC content, template quality, and assay complexity.
- Choose a safety margin if you want a more conservative hold.
- Set cycle count, final extension multiplier, and minimum final extension.
- Pick the rounding rule that matches your lab workflow.
- Press calculate to show the result above the form.
- Download the result as CSV or PDF if needed.
Example Data Table
| Amplicon | Enzyme Speed | GC % | Cycles | Recommended Per Cycle | Final Extension |
|---|---|---|---|---|---|
| 750 bp | 1000 bp/min | 48% | 30 | 50 sec | 5 min |
| 1.5 kb | 1500 bp/min | 58% | 35 | 1 min 15 sec | 6 min 15 sec |
| 3.2 kb | 600 bp/min | 66% | 35 | 7 min 45 sec | 38 min 45 sec |
FAQs
1) What does elongation time mean in PCR?
Elongation time is the hold during which the polymerase extends primers and builds the target strand. Longer targets usually need longer extension periods to finish synthesis reliably.
2) Why does amplicon length affect the result?
Polymerases synthesize DNA at finite speeds. A longer amplicon simply requires more time for the enzyme to move across the full target sequence.
3) Should GC-rich targets use longer extension times?
Often yes. GC-rich regions can be harder to amplify cleanly, so labs commonly allow extra extension time or use specialized enzymes and additives.
4) Is final extension the same as per-cycle elongation?
No. Final extension is a separate hold after the main cycles. It is usually longer and helps complete partially extended strands before the run ends.
5) Can a faster polymerase reduce total run time?
Yes. Higher extension speed lowers the base elongation requirement, which can shorten each cycle and reduce the total elongation-stage time.
6) Why round the time to 5 or 10 seconds?
Rounded values are easier to program on thermal cyclers and keep protocols consistent. Many labs prefer practical, repeatable setpoints instead of awkward decimal durations.
7) Does poor template quality change the estimate?
Yes. Degraded or impure template can slow efficient amplification, so adding a modest buffer can improve the chance of complete product formation.
8) Can this calculator help with qPCR or endpoint PCR?
It can provide a planning estimate for both, but always compare the result with your enzyme manufacturer guidance and your validated laboratory protocol.