From fce278c7b32d2fa554b7a640af6ca80281f4b632 Mon Sep 17 00:00:00 2001 From: Ernie Pedapati Date: Tue, 10 Mar 2026 12:49:48 -0400 Subject: [PATCH] Create sop: Standard Operating Procedure for Basic Polymerase Chain Reaction (PCR) --- ...sic-Polymerase-Chain-Reaction-Procedure.md | 250 ++++++++++++++++++ 1 file changed, 250 insertions(+) create mode 100644 SOPs/SOP-LAB-001-Basic-Polymerase-Chain-Reaction-Procedure.md diff --git a/SOPs/SOP-LAB-001-Basic-Polymerase-Chain-Reaction-Procedure.md b/SOPs/SOP-LAB-001-Basic-Polymerase-Chain-Reaction-Procedure.md new file mode 100644 index 0000000..9ee8dc8 --- /dev/null +++ b/SOPs/SOP-LAB-001-Basic-Polymerase-Chain-Reaction-Procedure.md @@ -0,0 +1,250 @@ +--- +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 | | | \ No newline at end of file