DNA Extraction Notes

Objectives

  • Rapid DNA extraction from environmental samples.
  • Set up PCR reactions for extracted DNA and proper controls.

DNA Barcoding Workflow

  • Collection of Samples: Gather samples from organisms.
  • DNA Extraction: Extract DNA from collected samples.
  • PCR Amplification: Use PCR to make billions of copies of barcoding regions.
  • Electrophoresis: Determine presence of PCR amplicons.
    • If absent, repeat specimen collection and extraction.
  • Next Steps: Last week, gel electrophoresis to check if we have amplicons for sequencing submission.

Sample Photography and DNA Extraction

  • DNA Location in Cells:

    • Eukaryotic cells: DNA in nucleus, mitochondria, chloroplasts (if present).

  • DNA Extraction Process:

    • Separate DNA from cellular components: cell walls, proteins, membranes.

    • Two protocols for DNA extraction:

    • Plants and Fungi Protocol:

      1. Mechanical abrasion used to break cells.
      2. Detergent and salt dissolve membranes and dissociate proteins.
      3. Centrifuge to discard tissue fragments.
      4. Precipitate DNA using isopropanol.
      5. Wash DNA pellet with ethanol; resuspend in smaller volume.

    • Invertebrate and Fish Protocol:

      1. Mechanical abrasion used for cell lysis.
      2. Alkaline lysis (NaOH) to denature DNA and proteins.
      3. Heat sample to speed up lysis.
      4. Add neutralizing buffer and centrifuge to separate proteins.
      5. DNA remains in supernatant without precipitation.

  • Crude Preps: Resulting solutions contain DNA and other cellular components but can be used for PCR.

  • Photography: Document samples with scale (ruler) for later reference.

Instructions for Capturing Images

  1. Open MicroCapture Pro Software.
  2. Focus and adjust image for scale:
    • Height, Zoom, Focus controls.
  3. Capture images and transfer to lab notebook:
    • Right-click to Copy, paste into Canvas.
  4. Delete images after transferring.

DNA Extraction Protocol for Plants and Fungi

  • Ingredients:
    • Extraction Buffer: 200 mM Tris, pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS.
  • Procedure Steps:
    1. Cut 2-3 mm³ piece of tissue into microcentrifuge tube (labelled).
    2. Grind with sterile pestle.
    3. Add Extraction Buffer and continue grinding.
    4. Vortex tube for 5 sec, centrifuge for 3 min.
    5. Transfer 300 µL of supernatant to new tube.
    6. Precipitate DNA with isopropanol and centrifuge.
    7. Wash pellet with 70% ethanol and centrifuge again.
    8. Resuspend DNA pellet in TE buffer (10 mM Tris, pH 8, 1 mM EDTA).

Alkaline Lysis DNA Extraction Protocol for Invertebrates and Fish

  • Ingredients:
    • Denature Buffer: 0.2 M NaOH, 0.2 mM EDTA.
    • Neutralization Buffer: 40 mM Tris pH 5.5.
  • Procedure Steps:
    1. Cut specimen piece and add to microcentrifuge tube with grinding sand.
    2. Add Denature Buffer and grind thoroughly.
    3. Centrifuge at maximum speed for a few seconds.
    4. Heat block for 10 min.
    5. Add Neutralization Buffer and mix.
    6. Transfer the top 50 µL supernatant to a new tube (DNA sample).

Polymerase Chain Reaction (PCR)

  • Master Mix Preparation:

    1. Adjust the number of reactions based on group size.
    2. Include negative control (H₂O) and positive control (instructor-provided DNA).
    3. Use Excel Master Mix Calculator to determine reactant volumes.
    4. Mix ingredients on ice using microcentrifuge tubes.

  • PCR Setup Procedure:

    1. List samples for amplification and control reactions.
    2. Identify major ingredients in PCR and their roles:
    • DNA template, primers, nucleotides, DNA polymerase.
    1. Thaw, vortex, and prepare primers before use.
    2. Keep solutions on ice until ready.
    3. Centrifuge samples to mix.
    4. Load PCR machine according to instructor's directions.

PCR Cycling Conditions

  • For plants, invertebrates, fish:

    • Denaturation: 94˚C for 30 sec
    • Annealing: 54˚C for 30 sec
    • Extension: 72˚C for 90 sec
    • Repeat for 34 cycles.

  • For fungi:

    • Denaturation: 94˚C for 30 sec
    • Annealing: 55˚C for 60 sec
    • Extension: 72˚C for 2 min
    • Repeat for 34 cycles.

Canvas Lab Notebook Entry

  1. Title and Date.
  2. Taxon of sample used for extraction.
  3. Photos of samples used (labeled with student names).
  4. Name of primers used.
  5. List of samples per PCR tube.
  6. PCR cycling conditions.
  7. Screenshot of Master Mix Worksheet.