Gel Electrophoresis II and DNA Sequencing Notes

Objectives

  • Run gels on PCR amplified samples

  • Identify successful PCR amplification samples

  • Prepare selected amplified samples for DNA sequencing

DNA Barcoding Workflow

  • Collection: Gather organism samples to extract DNA.

  • PCR Amplification: Amplify DNA barcoding regions, creating billions of copies.

  • Electrophoresis: Use gel electrophoresis to check for PCR amplicons.

    • If absent, repeat DNA extractions and PCR.

  • Results Utilization: Evaluate positive/negative controls before sequencing.

I. Agarose Gel Electrophoresis

  • Purpose of Agarose Gel:

    • Separates DNA fragments based on size.

    • Confirms presence and size of PCR amplicons by comparing to a DNA ladder.

  • Key Steps:

    1. Load PCR samples into lanes of the gel.

    2. Use DNA ladder in one lane to estimate amplicon sizes.

II. Gel Preparation

  • How Many Gel Boxes: Coordinate sample loading, ensuring positive/negative controls are included.

III. Preparing a 1% Agarose Gel

  1. Set up gel pouring tray as instructed.

  2. Insert gel comb to create wells.

  3. Pour melted agarose (~0.5 cm thick), ensuring caution (55ºC).

  4. Allow gel to solidify for 20-30 minutes.

IV. Sample Preparation & Loading

  1. Clean work surfaces with Mr. Clean.

  2. Thaw PCR products, vortex, and centrifuge briefly.

  3. Label new PCR tubes matching original samples.

  4. Add 8 µL PCR product to new tubes using fresh tips for accuracy.

  5. Add 2 µL 5X gel loading dye to each sample.

  6. Vortex and centrifuge briefly again.

  7. Prepare the gel box with 1X TAE buffer, ensuring the gel is submerged.

  8. Load 10 µL of DNA marker into the appropriate lane and then add samples.

Gel Imaging

  • Staining: Use GelRed stain, which fluoresces under UV light, facilitating visualization of DNA.

    • Handle with care; wear gloves to avoid contamination.

  • Imaging Process:

    1. Transfer gel to staining tray with GelRed for 15 min.

    2. Use the Bio-Rad GelDocEz to capture and save gel images.

C. Annotation & Interpretation

  1. Annotate gel images with lane information, including sample identity and size markers.

  2. Identify amplification results:

    • Check for positive control results to validate procedure integrity.

  3. Document results in the lab notebook using a results table:

    • Lane numbers, sample identities, presence of bands, and expected results.

D. Lab Notebook Requirements

  • Include the following:

    1. Title and date of the experiment.

    2. Annotated gel image with primer names and lane information.

    3. Brief description of amplification success.

    4. Conclusions about PCR performance and potential sequencing.

DNA Sequencing

  • DNA Barcode: The sequence (500+ base pairs) needed for taxonomic identification.

  • Sanger Sequencing Method:

    1. Uses ddNTPs to terminate DNA strand synthesis.

    2. Generates DNA fragments of various lengths for analysis.

    3. Fragments are separated via gel electrophoresis and analyzed to determine sequences.

Sample Prep for Sanger Sequencing

  1. Clean surfaces and wear protective gear.

  2. Confirm sample identity and mark the tube.

  3. Prepare labeled sequencing tubes and input PCR DNA:

    • Add 8 µL PCR DNA to each tube.

    • Add forward and reverse primers correctly to respective tubes.

  4. Record tube information in the sequencing spreadsheet to ensure proper tracking.

  5. Final submission of samples for Sanger sequencing.

Post-Lab Activities

  • Create a DNA Subway account for analysis.

  • Document usernames and sequencing tube details in lab notebook for submission.