Dundalk Institute of Technology 13

Introduction to Gel Preparation

  • Purpose: This section focuses on preparing and understanding the components involved in gel electrophoresis, specifically DNA visualization.

CyberSafe

  • Function: CyberSafe is added to the gel shortly after it has cooled to allow for visualization of DNA under UV light.

  • Binding: Binds to DNA, fluorescing to indicate the presence and separation of DNA.

  • Concentration:

    • Used in the preparation of 1x solution from a 10x stock solution:

      • Example ratio mentioned: 75 grams of gel requires a specific amount of CyberSafe, calculated from the 10x to 1x dilution.

TriTrac (Loading Buffer)

  • Role in Experiment:

    • Visualization Support: Although not visible with UV light, it tracks the progress of DNA through the gel.

    • Weighting Agent: Contains components (like glycine) that help weigh down DNA samples into the wells, preventing cross-contamination.

  • Concentration Explanation:

    • Generally, a 1 in 5 dilution is suggested for TriTrac:

      • Example: 1 uL of TriTrac for every 5 uL of sample.

DNA Ladder

  • Purpose: Used as a DNA marker, enabling estimation of DNA band sizes during electrophoresis.

  • Placement: Should be placed in one of the wells alongside the DNA samples to facilitate comparison.

Preparation Steps

  • Gel Cooling: After gel preparation, allow it to cool down before adding CyberSafe.

  • Adding Components:

    • After swirling the gel with the CyberSafe, ensure even distribution for fluorescence.

  • Calculation Reminder: Important to double-check the amount of each component (CyberSafe and TriTrac) required for the total gel volume (75 mL in this example).

Key Takeaways

  • Importance of CyberSafe: Forgetting to add CyberSafe means the DNA can separate but cannot be visualized, which is counterproductive to the experiment.

  • TriTrac and DNA Ladder: Both play crucial roles in ensuring that DNA samples can be monitored and accurately sized during the process.