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
- Weigh 1 g agarose for a 1% gel in 100 mL 1X TAE buffer (adjust for volume/concentration as needed).
- Microwave gently until agarose dissolves completely (clear solution).
- Cool to ~55°C, add DNA stain as per manufacturer’s instructions.
- Pour gel into casting tray with comb in place. Allow to solidify (20–30 minutes).
5.3 Sample Preparation
- Mix DNA samples (2–5 µL) with 6X loading dye (1 µL).
- Centrifuge briefly to collect contents at the bottom of the tube.
5.4 Gel Electrophoresis
- Place the solidified gel into the electrophoresis tank and cover with 1X TAE buffer.
- Carefully load samples and DNA ladder into wells.
- Connect electrodes (black to negative, red to positive) and run at 80–120 V for 45–60 minutes.
5.5 Visualization and Documentation
- Turn off power, remove gel, and visualize under UV or blue-light transilluminator.
- Capture gel image with gel documentation system.
- Label image with gel number, date, DNA IDs, ladder type, and voltage used.
- Attach image to Gel Electrophoresis Log (Annexure-1).
5.6 Cleanup and Waste Disposal
- Dispose of used gels and staining agents per biosafety waste guidelines.
- 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
- Gel Electrophoresis Log (Annexure-1)
- 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 |