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전체 프로토콜 분야

PCR & Quantitative Amplification

내 assay에 맞춰 완성하는 PCR 프로토콜

실행 순서와 합리적인 시작 조건은 준비해 두었습니다. 내 표적의 primer·probe와 실제 kit 조건을 채우고, Word 또는 Excel로 저장해 실험에 사용하세요.

편집 가능한 시작 프로토콜

10 개

이 프로토콜은 무엇을 하나요?

증폭 방식과 assay 입력을 먼저 선택하고 primer·probe, master mix, control, cycling과 결과 판정 항목을 해당 방식에 맞게 구성합니다.

지원하는 증폭 방식

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

적용 경계

사용자의 실제 표적 서열과 kit·장비 조건을 자동으로 확정하지 않습니다. MIQE·dMIQE와 제조사 지침은 설계 및 보고 기준으로 구분합니다.

SP-PCR-001Conventional PCR

Routine endpoint PCR — Taq-based 25 µL starting protocol

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

공식 가이드 검토됨
ORDERED_STEPSTARGET_SPECIFIC_OLIGOS_REQUIREDMANUFACTURER_IFU_OVERRIDESCONTROL_GATED

먼저 내 assay 정보를 채워 주세요

실행 순서와 합리적인 시작 조건을 정리한 초안입니다. 샘플 primer·probe 서열은 넣지 않았습니다. 반드시 자신의 표적 서열을 입력하고, 실제 효소·kit·장비 설명서와 assay 검증 결과에 맞게 조건을 확정하세요.

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.

조건: 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.

조건: 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.

온도: On ice or as directed by the selected master mix시약: 12.5 µL 2× Taq master mix; 0.5 µL each 10 µM primer; water; template added separately조건: 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.

조건: 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.

시간: 30 seconds온도: 94–95°C조건: 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.

시간: 15–30 seconds온도: 94–95°C조건: 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.

시간: 15–60 seconds온도: Start about 5°C below the lower primer Tm조건: 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.

시간: About 1 minute per kb온도: 68–72°C조건: 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.

시간: 5 minutes; then hold온도: 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.

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