Standard Operating Procedure: Agarose Gel Electrophoresis

This Standard Operating Procedure (SOP) outlines the standardized process for performing agarose gel electrophoresis, a fundamental technique used to separate DNA or RNA fragments by size. Consistent adherence to these protocols is critical to ensure high-resolution banding patterns, minimize the risk of sample contamination, and maintain laboratory safety. This procedure applies to all personnel utilizing the electrophoresis equipment within this facility.

Phase 1: Gel Preparation

• Determine the required agarose percentage based on the size of the DNA fragments (typically 0.8% for large fragments, 1.5–2.0% for small fragments).
• Measure the appropriate amount of agarose powder and add it to a heat-resistant flask containing the specified volume of 1X TAE or TBE buffer.
• Microwave the mixture in short pulses, swirling gently until the agarose is completely dissolved and the solution appears clear.
• Allow the solution to cool to approximately 50–60°C before adding the DNA stain (e.g., SYBR Safe) according to manufacturer specifications.
• Secure the casting tray with gates and insert the appropriate combs.
• Pour the agarose solution into the tray, ensuring no air bubbles are present; allow the gel to solidify completely (approx. 30–45 minutes).

Phase 2: Sample Preparation and Loading

• Prepare DNA samples by mixing them with the appropriate loading dye (typically 1X–5X concentration).
• Carefully remove the gel combs once the gel is fully solidified.
• Place the gel into the electrophoresis tank and submerge it in the same running buffer (TAE or TBE) used to prepare the gel.
• Load the molecular weight ladder (marker) into the first well, ensuring the volume matches the experimental samples.
• Carefully pipette the DNA/dye mixture into subsequent wells, using a steady hand to avoid puncturing the well floor.
• Record the loading order in the laboratory notebook or digital tracking system.

Phase 3: Electrophoresis and Visualization

• Secure the tank lid and connect the leads to the power supply, ensuring the red (positive) and black (negative) cables are correctly matched (DNA travels toward the positive electrode).
• Set the voltage according to the gel percentage and fragment size (standard: 80V–120V); do not exceed 5V/cm of gel length.
• Monitor the migration of the dye front; terminate the run before the dye front reaches the end of the gel.
• Carefully remove the gel from the tank and transfer it to a clean imaging system (e.g., UV or Blue Light transilluminator).
• Capture the image and adjust exposure settings to ensure optimal band visibility without pixel saturation.

Pro Tips & Pitfalls

• Pitfall: Smiling Bands. Running the gel at too high a voltage causes the gel to heat up, distorting the migration pattern. If bands appear "smiled," decrease the voltage.
• Pro Tip: Bubble Management. If bubbles appear while pouring, use a clean pipette tip to pop them or push them to the side before the gel sets; bubbles in the well area will cause the current to divert, resulting in distorted bands.
• Pitfall: Buffer Exhaustion. Using the same buffer for too many runs will lead to a change in pH and poor migration. Change the running buffer every 3–4 runs to maintain consistent results.
• Pro Tip: Well Integrity. When removing the comb, do so slowly and vertically. Pulling at an angle often tears the delicate agarose wells.

Frequently Asked Questions (FAQ)

1. Why is my DNA band smeared instead of a sharp, clear band? Smearing usually indicates that the DNA sample is degraded, the sample load was too high (overloading), or the gel was run at too high a voltage. Ensure samples are stored correctly and try decreasing the amount of DNA loaded.

2. Can I reuse the electrophoresis running buffer? Yes, but with limitations. Buffer can be reused for a few runs, but it loses its buffering capacity over time due to electrolysis. If you notice the dye front migrating abnormally or inconsistent band separation, replace the buffer immediately.

3. What should I do if my DNA didn't migrate at all? First, check that the leads are connected to the correct terminals (Black to Negative, Red to Positive). If the connections are correct, verify that the power supply is functional and that the buffer in the tank is high enough to create a complete circuit.