SOPs/SOP-LAB-001-Basic-Polymerase-Chain-Reaction-Procedure.md

---
title: "Standard Operating Procedure for Basic Polymerase Chain Reaction (PCR)"
author: ""
date: 2026-03-10
version: "1.0"
status: "Draft"
---

## 1. Purpose

The purpose of this Standard Operating Procedure (SOP) is to define the standardized method for performing a basic Polymerase Chain Reaction (PCR) for DNA amplification in a controlled laboratory environment.  

This procedure ensures:
- Consistent and reproducible PCR results  
- Compliance with ISO 9001 and ISO 13485 quality management requirements  
- Data integrity in accordance with FDA 21 CFR Part 11 (where electronic systems are used)

---

## 2. Scope

This SOP applies to all laboratory technicians performing basic PCR procedures within the laboratory facility.

This procedure covers:
- Preparation of reagents and master mix  
- Sample handling  
- Thermal cycler setup  
- Amplification process  
- Post-PCR handling  
- Documentation and data recording  

This SOP does not cover:
- Quantitative PCR (qPCR)  
- Reverse transcription PCR (RT-PCR)  
- Advanced assay validation  

---

## 3. References

- ISO 9001: Quality Management Systems – Requirements  
- ISO 13485: Medical Devices – Quality Management Systems  
- FDA 21 CFR Part 11 – Electronic Records and Electronic Signatures  
- Laboratory Biosafety Manual ""  
- Equipment Manual: Thermal Cycler Model ""  

---

## 4. Definitions

| Term | Definition |
|------|------------|
| PCR | Polymerase Chain Reaction, a method used to amplify DNA sequences |
| Master Mix | A premixed solution containing DNA polymerase, dNTPs, buffer, and MgCl₂ |
| Template DNA | DNA sample containing the target sequence |
| NTC | No Template Control |
| Thermal Cycler | Instrument used to automate PCR temperature cycling |

---

## 5. Roles and Responsibilities

| Role | Responsibility |
|------|---------------|
| Laboratory Technician | Perform PCR according to this SOP and document all activities |
| Laboratory Supervisor | Ensure training, review records, and approve deviations |
| Quality Assurance | Ensure compliance with QMS and regulatory requirements |

---

## 6. Safety and Environmental Considerations

- Wear appropriate PPE: lab coat, gloves, and eye protection.
- Handle biological samples in accordance with biosafety guidelines.
- Use aerosol-resistant pipette tips.
- Dispose of biological and chemical waste according to laboratory waste procedures "".
- Avoid cross-contamination by maintaining separate pre- and post-PCR areas.

---

## 7. Equipment and Materials

### 7.1 Equipment

| Equipment | Model | ID No. | Calibration Due Date |
|-----------|-------|--------|----------------------|
| Thermal Cycler | "" | "" | "" |
| Microcentrifuge | "" | "" | "" |
| Micropipettes | "" | "" | "" |
| Vortex Mixer | "" | "" | "" |

### 7.2 Reagents and Consumables

| Item | Manufacturer | Lot No. | Expiry Date |
|------|-------------|----------|------------|
| PCR Master Mix | "" | "" | "" |
| Forward Primer | "" | "" | "" |
| Reverse Primer | "" | "" | "" |
| Template DNA | "" | "" | "" |
| Nuclease-Free Water | "" | "" | "" |
| PCR Tubes/Plates | "" | "" | "" |

---

## 8. Procedure

### 8.1 Pre-Procedure Checks

- Verify equipment calibration status.
- Confirm reagent integrity and expiration dates.
- Thaw reagents on ice.
- Prepare a clean PCR workstation.
- Record reagent lot numbers in the PCR worksheet.

---

### 8.2 Preparation of Master Mix

1. Calculate total reaction volume and number of reactions, including:
   - Test samples  
   - Positive control  
   - NTC  
   - 10% excess volume to account for pipetting error  

2. Prepare master mix according to assay design:

| Component | Volume per Reaction (µL) | Final Concentration |
|------------|--------------------------|---------------------|
| Master Mix | "" | "" |
| Forward Primer | "" | "" |
| Reverse Primer | "" | "" |
| Nuclease-Free Water | "" | "" |

3. Mix gently by pipetting or brief vortex.
4. Centrifuge briefly to collect contents.

---

### 8.3 Reaction Setup

1. Label PCR tubes clearly.
2. Aliquot appropriate volume of master mix into each tube.
3. Add template DNA to designated tubes.
4. Add nuclease-free water to NTC.
5. Cap tubes securely.
6. Briefly centrifuge to remove air bubbles.

---

### 8.4 Thermal Cycler Programming

Program the thermal cycler as follows:

| Step | Temperature (°C) | Time | Cycles |
|------|------------------|------|--------|
| Initial Denaturation | "" | "" | 1 |
| Denaturation | "" | "" | "" |
| Annealing | "" | "" | "" |
| Extension | "" | "" | "" |
| Final Extension | "" | "" | 1 |
| Hold | "" | "" | 1 |

- Verify correct program selection before starting.
- Record program name and run ID in the PCR worksheet.

---

### 8.5 PCR Run

- Place tubes in thermal cycler.
- Close lid securely.
- Start run and confirm program initiation.
- Record run start and end times.

---

### 8.6 Post-PCR Handling

- Remove tubes after completion.
- Store amplified products at "" °C if required.
- Proceed to downstream analysis if applicable (e.g., gel electrophoresis).
- Decontaminate work surfaces.

---

## 9. Quality Control

- Include positive control and NTC in each run.
- Acceptable result criteria:

| Control | Expected Result | Acceptance Criteria |
|----------|----------------|--------------------|
| Positive Control | Amplification observed | Clear expected band |
| NTC | No amplification | No visible band |

- Document deviations and notify supervisor if acceptance criteria are not met.
- Initiate Nonconformance Report (NCR) if required.

---

## 10. Documentation and Records

The following records must be completed:

- PCR Worksheet ""  
- Equipment Logbook  
- Reagent Log  
- Deviation Report (if applicable)  

For electronic records:
- Ensure user access control is maintained.
- Electronic signatures must comply with FDA 21 CFR Part 11.
- Audit trails must be enabled where applicable.

Records retention period: "" years.

---

## 11. Deviations and Corrective Actions

- Any deviation from this SOP must be documented.
- Notify Laboratory Supervisor immediately.
- Perform root cause investigation if required.
- Implement corrective and preventive actions (CAPA) per procedure "".

---

## 12. Training Requirements

- Personnel must be trained on this SOP prior to performing PCR independently.
- Training records must be maintained.
- Competency assessment frequency: "".

---

## 13. Change History

| Version | Date | Description of Change | Author |
|----------|------------|----------------------|--------|
| 1.0 | 2026-03-10 | Initial draft | "" |

---

## 14. Approval Signatures

| Name | Title | Signature | Date |
|------|-------|------------|------|
| "" | Laboratory Supervisor |  |  |
| "" | Quality Assurance |  |  |
| "" | Laboratory Manager |  |  |