Electroforesis en gel de poliacrilamida | | UPV

Electrophoresis Sample Loading Process

Preparing the System

• To begin loading samples, add electrophoresis buffer into the cavity formed between the gels until it reaches the top. Remove the comb carefully to avoid breaking wells.

• Wait briefly to ensure that the interior cavity is sealed and that the buffer does not drop. Continue adding buffer until it overflows, ensuring both upper and lower parts of the gel are covered.

Ensuring Circuit Integrity

• Confirm that the buffer level remains above a specific line on the smaller gel to maintain a closed circuit before proceeding with sample preparation.

Loading Samples

• Load samples into wells by using a pipette positioned towards you, submerging it in buffer while touching the higher glass, then descending to touch the smaller glass without applying force.

• Gently release each sample from the pipette tip into its respective well, ensuring no bubbles form or escape from within.

Handling Bubbles and Sample Volume

• Be cautious of air bubbles when loading samples, especially since loading buffer contains SDS which can create bubbles. Release any trapped air before allowing sample flow.

• Adjust pipette volume appropriately to prevent air from entering; if necessary, keep pressure on plunger while reintroducing it into the cavity for accurate sample delivery.

Observing Color Changes

• Note any color changes in samples due to pH shifts as they are loaded; this indicates successful handling and proper technique.

Finalizing Sample Loading

• For control samples like PSA (at 0.5 mg/mL), calculate required volumes accurately (e.g., 20 µL for 10 µg).

• Ensure no bubbles during final sample loading; position pipette correctly and release slowly to avoid splashing or bouncing against well walls.

Completing Setup

• After all samples are loaded successfully, cover the chamber with electrodes (red and black), ensuring they fit snugly into place for optimal performance during electrophoresis.