Standard Operating Procedure for Agarose Gel Electrophoresis in Biosimilar R&D

Department	Biosimilars
SOP No.	SOP/BS/008/2025
Supersedes	SOP/BS/008/2022
Page No.	Page 1 of 10
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To establish a standardized procedure for performing agarose gel electrophoresis to separate, identify, and analyze DNA fragments for plasmid confirmation and molecular biology workflows in biosimilar development.

2. Scope

This SOP applies to all R&D personnel involved in analyzing plasmid DNA, PCR products, and restriction digests through agarose gel electrophoresis within the Biosimilars department.

3. Responsibilities

• Research Assistant: Prepares gel, loads samples, runs electrophoresis, and documents results.
• Lab Technician: Maintains electrophoresis equipment and ensures buffer/media quality.
• QA Representative: Reviews gel documentation for completeness and compliance.

4. Accountability

The Head of Molecular Biology is accountable for ensuring that electrophoresis procedures are performed safely, reproducibly, and in accordance with molecular characterization protocols.

5. Procedure

5.1 Materials Required

• Agarose powder
• 1X TAE or TBE buffer
• DNA ladder (100 bp or 1 kb)
• DNA samples (e.g., plasmids, PCR products)
• 6X Loading dye
• DNA stain (e.g., Ethidium Bromide, SYBR Safe)
• Gel casting tray and comb, electrophoresis tank, power supply

5.2 Gel Preparation

1. Weigh 1 g agarose for a 1% gel in 100 mL 1X TAE buffer (adjust for volume/concentration as needed).
2. Microwave gently until agarose dissolves completely (clear solution).
3. Cool to ~55°C, add DNA stain as per manufacturer’s instructions.
4. Pour gel into casting tray with comb in place. Allow to solidify (20–30 minutes).

5.3 Sample Preparation

1. Mix DNA samples (2–5 µL) with 6X loading dye (1 µL).
2. Centrifuge briefly to collect contents at the bottom of the tube.

5.4 Gel Electrophoresis

1. Place the solidified gel into the electrophoresis tank and cover with 1X TAE buffer.
2. Carefully load samples and DNA ladder into wells.
3. Connect electrodes (black to negative, red to positive) and run at 80–120 V for 45–60 minutes.

5.5 Visualization and Documentation

1. Turn off power, remove gel, and visualize under UV or blue-light transilluminator.
2. Capture gel image with gel documentation system.
3. Label image with gel number, date, DNA IDs, ladder type, and voltage used.
4. Attach image to Gel Electrophoresis Log (Annexure-1).

5.6 Cleanup and Waste Disposal

1. Dispose of used gels and staining agents per biosafety waste guidelines.
2. Wipe down the tank and tray using 70% ethanol or suitable lab disinfectant.

6. Abbreviations

• SOP: Standard Operating Procedure
• TAE: Tris-Acetate-EDTA
• TBE: Tris-Borate-EDTA
• QA: Quality Assurance

7. Documents

1. Gel Electrophoresis Log (Annexure-1)
2. Gel Documentation Record (Annexure-2)

8. References

• ICH Q5B – Analysis of Expression Constructs
• WHO Guidelines on Molecular Techniques for DNA Products
• CDSCO Manual on Molecular Biology Practices

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Gel Electrophoresis Log

Date	Gel ID	Samples Loaded	Ladder	Voltage	Run Time	Operator
03/05/2025	AG-058	pUC19, PCR-A, PCR-B	1 kb Plus	100 V	45 min	Sunita Reddy

Annexure-2: Gel Documentation Record

Image File Name	Sample IDs	Date Captured	Remarks
gel_080525.png	pUC19, PCR-A	03/05/2025	Sharp bands, expected sizes

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated electrophoresis voltage range and gel image tracking	Annual update