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PCR & Quantitative Amplification

Complete a PCR protocol for your own assay

The ordered workflow and rational starting conditions are ready. Add your target-specific primers or probe, align the settings with your actual kit, then save the working protocol as Word or Excel.

Editable starter protocols

10 protocols

What do these protocols do?

They select the amplification mode and assay input first, then organize primer or probe, master mix, controls, cycling, and result-review fields for that mode.

Supported amplification modes

  • • Conventional PCR
  • • qPCR / Real-time PCR
  • • RT-PCR
  • • RT-qPCR
  • • dPCR / ddPCR

Scope boundary

They do not infer the user’s target sequence or finalize kit and instrument conditions. MIQE, dMIQE, and manufacturer instructions retain separate roles.

SP-PCR-001Conventional PCR

Routine endpoint PCR — Taq-based 25 µL starting protocol

PCR & Quantitative Amplification · CURATED-SP-PCR-001-V1

Official guidance reviewed
ORDERED_STEPSTARGET_SPECIFIC_OLIGOS_REQUIREDMANUFACTURER_IFU_OVERRIDESCONTROL_GATED

Add your assay information before use

This draft provides an ordered workflow and rational starting conditions. It intentionally contains no sample primer or probe sequence. Enter your target-specific oligonucleotides and finalize every condition against the actual enzyme, kit, instrument instructions, and assay-validation data.

1

Define the target and primers

Record the target accession or genomic region, expected amplicon size, and a validated target-specific primer pair. This starter intentionally supplies no primer sequence.

Condition: USER REQUIRED: enter forward and reverse sequences; verify specificity, orientation, and melting temperatures before setup.
2

Create the tube map and controls

Assign samples and controls before opening reagents so that every result can be interpreted against contamination and amplification controls.

Condition: Include an NTC and target-positive control; add an extraction blank when samples passed through extraction.
3

Prepare the master mix

Prepare enough primer-containing master mix for all reactions plus pipetting overage. Add template separately.

Temperature: On ice or as directed by the selected master mixReagent: 12.5 µL 2× Taq master mix; 0.5 µL each 10 µM primer; water; template added separatelyCondition: 25 µL final; 1× mix; 0.2 µM each primer; prepare 10% overage.
4

Aliquot and add template

Dispense master mix, add the planned template volume to each sample, replace template with nuclease-free water in the NTC, then cap and briefly spin.

Condition: Use aerosol-resistant tips; keep post-PCR material out of the setup area.
5

Initial denaturation

Denature the input template using the starting condition for a routine Taq reaction.

Duration: 30 secondsTemperature: 94–95°CCondition: Use a longer initial denaturation only when required by the template or polymerase IFU.
6

Cycle — denaturation

Denature amplified DNA at the start of each cycle.

Duration: 15–30 secondsTemperature: 94–95°CCondition: 25–35 cycles total; choose cycle number from target abundance and validation data.
7

Cycle — primer annealing

Anneal the validated primer pair. Optimize with a gradient when the assay is new or specificity is poor.

Duration: 15–60 secondsTemperature: Start about 5°C below the lower primer TmCondition: Do not use a fixed annealing temperature before primer Tm and specificity are known.
8

Cycle — extension

Extend the amplicon using the temperature and rate required by the selected polymerase.

Duration: About 1 minute per kbTemperature: 68–72°CCondition: Use the enzyme IFU when it differs from this Taq starting point.
9

Final extension and hold

Complete partially extended products, then hold the reactions until collection.

Duration: 5 minutes; then holdTemperature: 68–72°C; then 4–10°C
10

Verify the endpoint product

Resolve an appropriate aliquot with a size standard and confirm a product at the expected size. Interpret samples only when controls behave as planned.

Condition: Do not accept the run if the NTC contains a target-sized product or the positive control fails.