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Suggested Protocols

探索型MVP
すべてのプロトコル分野

PCR & Quantitative Amplification

自分のassayに合わせてPCR protocolを完成

実行順序と合理的な開始条件は準備済みです。標的固有のprimer・probeと実際のkit条件を入力し、WordまたはExcelに保存して使用できます。

編集可能な開始プロトコル

10 件

このプロトコルは何を行いますか？

増幅方式とassay inputを先に選び、primer・probe、master mix、control、cycling、結果判定をその方式に合わせて構成します。

対応する増幅方式

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

適用範囲

利用者の標的配列やkit・装置条件を自動確定しません。MIQE・dMIQEとメーカー指示は設計・報告基準として区別します。

SP-PCR-001Conventional PCR

Routine endpoint PCR — Taq-based 25 µL starting protocol

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

公式ガイド 確認済み
ORDERED_STEPSTARGET_SPECIFIC_OLIGOS_REQUIREDMANUFACTURER_IFU_OVERRIDESCONTROL_GATED

使用前に自分のassay情報を入力してください

実行順序と合理的な開始条件を整理した下書きです。サンプルのprimer・probe配列は含めていません。必ず自分の標的配列を入力し、実際の酵素・kit・装置の説明書とassay検証データに合わせて条件を確定してください。

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.

条件: 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.

条件: 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.

温度: On ice or as directed by the selected master mix試薬: 12.5 µL 2× Taq master mix; 0.5 µL each 10 µM primer; water; template added separately条件: 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.

条件: 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.

時間: 30 seconds温度: 94–95°C条件: 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.

時間: 15–30 seconds温度: 94–95°C条件: 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.

時間: 15–60 seconds温度: Start about 5°C below the lower primer Tm条件: 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.

時間: About 1 minute per kb温度: 68–72°C条件: 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.

時間: 5 minutes; then hold温度: 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.

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