Gel Electrophoresis Notes

Gel Electrophoresis Objectives:

  • Analyze PCR products (amplicons) using agarose gel electrophoresis.

  • Use controls for interpreting experimental results.

  • Document results in a Lab Notebook entry.

DNA Barcoding Overview

  • DNA barcoding involves:

    • Collecting organism samples.

    • Extracting DNA from samples.

    • Using PCR to amplify DNA barcoding regions (billions of copies).

  • Goals of gel electrophoresis in this context:

    1. Confirm the success of DNA extraction.

    • Expected outcome: Student PCRs should not amplify if DNA extraction fails; positive control should amplify.

    1. Determine PCR success through agarose gel electrophoresis.

    • Use positive/negative controls for evaluation.

    • If successful, aliquot for sequencing; if not, redo DNA extractions and PCRs.

Agarose Gel Electrophoresis

  • Visualize PCR amplicons using agarose gel electrophoresis to separate and view DNA fragments based on size.

  • Key concepts:

    • DNA has a net negative charge and migrates towards the positive pole in an electric field.

    • Agarose is a medium that restricts larger DNA molecules more than smaller ones; smaller molecules migrate faster.

    • DNA conformation also affects migration rates.

  • Procedure:

    • Load PCR samples into the wells of the gel.

    • Use DNA markers to estimate PCR product sizes (measured in base pairs, or bp).

  • Base Pair Conversion:

    • $1000 ext{ bp} = 1 ext{ kb}$ (kilobase).

Preparing Agarose Gel

  • To prepare a 1% agarose gel:

    • $1 ext{ g of agarose per } 100 ext{ mL of TAE buffer}$.

    • TAE buffer components: 40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.3.

    • Heat with microwave until dissolved.

    • Cool to 55°C, pour into a gel tray, and let solidify for 20-30 min.

Preparing PCR Samples for Gel Electrophoresis

  • Clean workspace and wear gloves.

  • Thaw and vortex PCR products; centrifuge to mix.

  • Label new PCR tubes for transferring samples.

  • Add 8 µL of each PCR product to labeled tubes and 2 µL of 5X gel loading dye to each:

    • Loading dye increases sample density for sinking into wells.

    • It also allows tracking of electrophoresis progress (does not stain DNA).

  • Submerge gel in 1X TAE buffer before loading samples.

Electrophoresis Procedure

  1. Remove the comb from the gel and place the gel tray in the gel box.

  2. Fill the gel box with ~300 mL of 1X TAE buffer to submerge the gel.

  3. Record samples in lanes based on loading plan, ensuring to include a DNA marker lane.

  4. Set the voltage (130 V for 30 minutes) and monitor the progress of the dye.

Gel Imaging and Staining

  • Staining using GelRed which fluoresces under UV light when bound to DNA.

  • Handle GelRed with caution (it is non-toxic but handle with gloves).

  • Protocol:

    1. Incubate gel in GelRed stain for 15 minutes while gently swirling.

    2. Transfer gel to imaging plate and use UV imaging software to capture gel images.

    3. Save and annotate images with lane information and DNA marker sizes.

Annotation and Interpretation of Gel

  • Open Gel Image Template and delete existing image.

  • Insert gel image and annotate:

    • Crop to show only relevant lanes.

    • Adjust marker sizes for corresponding DNA fragments.

    • Document sampled taxa.

  • Interpret results based on PCR controls:

    1. If amplification occurs in negative control, contamination may have occurred.

    2. Rely on positive control for troubleshooting student samples.

    3. Assess whether PCR products are suitable for sequencing.

Submission and Reporting

  • Submit group’s Lab Notebook template by midnight of the lab day.

  • Prepare a Results and Discussion report for individual follow-up:

    1. Describe goals of the experiment and summary of observed results.

    2. Include annotated gel with size markers and sample information.

    3. Discuss predictions versus observed results and conclusions regarding sequencing submissions.