Dundalk Institute of Technology 22

  • Purpose of CyberSafe

    • CyberSafe is a fluorescent reagent used to visualize DNA when exposed to UV light after electrophoresis.

    • Can be utilized to help observe DNA bands during gel electrophoresis.

  • Principles of PCR (Polymerase Chain Reaction)

    • PCR is used to amplify specific DNA sequences.

    • Selects target DNA for amplification through the design of primers.

    • Primers are designed against specific DNA sequences, which ensures only desired sections are amplified.

  • Preparation of Agarose Gel

    • Pouring the gel:

      • Necessary to add a comb while pouring to create wells for loading samples.

      • Allows for the separation of samples in the gel.

    • Setting the gel:

      • Let the gel set for 15-20 minutes and iron out any bubbles that may affect sample loading.

  • Loading Samples into the Gel

    • Samples must be mixed with loading dye before being added to wells.

    • Purpose of Loading Buffer:

      • Glycerol in the loading dye ensures samples sink into wells and prevents cross-contamination.

      • TriTrack dye tracks DNA but does not visualize it; it indicates the progress during electrophoresis.

  • Running the Gel

    • Always include a DNA ladder alongside samples for size reference.

    • Expected size of amplified product: 732 base pairs

    • Gel Percentage:

      • Use a 1% gel for larger DNA fragments (like 732 bp) and a 2% gel for smaller fragments.

      • Higher agarose percentages tighten the mesh, which can trap larger DNA fragments.

  • Visualization of Bands

    • Allow for the orientation of DNA bands by counting and measuring their size against a DNA ladder for identification.

    • Important to include pertinent labels but avoid overcrowding.

  • PCR Details in Introduction

    • Include a background of the PCR technique used (e.g., colony PCR focused on pGlow plasmid).

    • Discuss the significance and applications of PCR in research and industry, ensuring that applications are explained with context.

  • Results Discussion

    • Analyze the presence/absence of DNA bands and their sizes to confirm the success of PCR.

    • Consider discussing the characteristics of primers and their effectiveness, evaluating attributes like GC content and length to ensure specificity.

  • Limitations and Controls

    • Discussing limitations of the experiment is crucial, including the absence of positive controls that could validate PCR results.

    • May touch on improvements for future experiments based on results or findings from the current experiment.

  • Conclusion

    • Summarize overall findings and implications of the experiment, reiterating the importance of controls, visualization techniques, and primer design.