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

title, author, date, version, status

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

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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

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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 ""

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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

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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

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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.

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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	""	""	""

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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.

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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.

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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.

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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.

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8.5 PCR Run

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

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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.

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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.

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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.

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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 "".

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12. Training Requirements

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

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13. Change History

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

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14. Approval Signatures

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