Agarose Gel Electrophoresis Notes

  • Agarose Gel Electrophoresis Overview

    • Common technique to separate DNA fragments by size.
    • Utilized in molecular biology and genetics research.
    • Cost-effective and straightforward method for DNA analysis.

  • Objectives of the Lab

    • Describe agarose gel and its role in separating different DNA fragment sizes.
    • Explain the function of electricity and TBE buffer in the electrophoresis process.
    • Load samples without damaging wells or losing material.
    • Run agarose gel and photograph DNA after electrophoresis.
    • Use a DNA ladder to label and estimate sizes of bands in the photo.

  • Introduction

    • Focus on PCR product to verify success of DNA extraction/PCR steps.
    • Use of agarose gel to analyze DNA through electric current migration.

  • Steps Involved in Agarose Gel Electrophoresis

    1. Casting the Gel:
    • Agarose, derived from seaweed, is mixed with buffering solution and heated.
    • Cools in a tray, creating a gel matrix with wells formed by a comb.
    1. Loading Samples:
    • Mix DNA samples with loading dye for density and visual tracking.
    • Load samples into gel wells using a micropipette cautiously.
    1. Electrophoresis:
    • Place gel in an electrophoresis apparatus, submerged in TBE buffer.
    • Apply electric current: DNA (negatively charged) moves towards positive electrode.
    • Small fragments travel faster than larger ones.
    1. Visualization:
    • DNA fragments are made visible using DNA dyes (e.g., ethidium bromide, SYBR Green).
    • Dyes intercalate with DNA and fluoresce under UV light.
    1. Analysis:
    • Compare migration distance of DNA fragments with a DNA ladder of known sizes to estimate fragment sizes.
    • Useful in applications like DNA fingerprinting, analyzing PCR, and RFLP analysis.

  • DNA Ladder:

    • A 100 bp DNA ladder from New England Biolabs (NEB), ranging from 100 bp to 1517 bp.
    • Contains a 6X gel loading dye mix to facilitate gel loading and visualization.

  • Gel Preparation & Loading Process

    • Two gels: one for practice (unstained) and one for actual samples (stained).
    • Carefully remove comb from the gel to avoid damaging wells.
    • Transfer gel into electrophoresis box, covering with 1X TBE buffer.

  • Buffer and Sample Preparation:

    • Prepare 100 bp DNA ladder with 8 µL dH2O, 1 µL ladder, and 3 µL gel loading dye.
    • Prepare PCR samples: 4 µL sterile distilled water, 5 µL PCR reaction, and 3 µL gel loading dye.
    • Ensure to record which sample is in each lane before running the gel.

  • Running the Gel:

    1. Load DNA ladder and samples into respective wells.
    2. Connect the gel box lid correctly, ensuring electrodes are oriented properly.
    3. Turn on the power supply, observing the migration of samples.
    4. Monitor results, should see satisfactory results within 15-30 minutes.
    5. Turn off power and view the gel under transilluminated blue light.

  • Cleanup Procedures:

    • Dispose of used tubes and solutions in designated containers.
    • Rinse and clean gel box.
    • Store extra PCR reactions on ice or as directed.

  • Technical Preparation and Equipment:

    • Equipment includes minigel apparatus, power supply, micropipettors, TBE buffer, 1.5 mL tubes, etc.
    • Ensure ample supplies for practice and running gels during lab sessions.