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Sample Protocol: DNA Extraction from Whole Blood Using High Pure Filter System

This sample demonstrates my ability to write clear and detailed molecular biology protocols. It follows a standard DNA extraction workflow using silica-column purification kits.

Purpose:
To isolate high-quality DNA from mammalian whole blood using silica-based spin columns and proteinase K digestion, suitable for downstream PCR applications.

Audience:
Clinical lab technicians and molecular biology researchers

Pre-Extraction Reagent Preparation

Proteinase K (Vial 3 – Pink Cap):

  • Add 4.5 ml double-distilled water.
  • Aliquot and store at –15°C to –25°C (stable for 12 months).
  • Label each aliquot tube (e.g., Eppendorf) with "ProtK", the preparation date, and the aliquot number (e.g., 1st, 2nd, etc.).

Inhibitor Removal Buffer (Vial 4a – Black Cap):

  • Add 20 ml absolute ethanol.
  • Label with preparation date.
  • Store at +15°C to +25°C.

Wash Buffer (Vial 4 – Blue Cap):

  • Add 80 ml absolute ethanol.
  • Label with preparation date.
  • Store at +15°C to +25°C.

Protocol: Isolation of Nucleic Acids from Whole Blood

Note: To avoid cross-contamination, perform this protocol on a clean lab bench, not inside biosafety cabinets used for pathogen work or sterile applications.
Tip: Always use counterweights when centrifuging.

  1. Prepare Elution Buffer

    • Warm to +70°C before use.

  2. Sample Input

    • If <200 μl of blood: top up to 200 μl with PBS.
    • If >200 μl (max 300 μl): adjust all volumes proportionally.

  3. Lysis Step

    • In a 1.5 ml nuclease-free microcentrifuge tube, add:
      • 200 μl of whole blood sample
      • 200 μl Binding Buffer
      • 40 μl reconstituted Proteinase K
    • Mix immediately.
    • Incubate at +70°C for 10 minutes.

  4. Precipitation

    • Add 100 μl isopropanol to the lysate.
    • Mix thoroughly.

  5. Binding Step

    • Insert a High Pure Filter Tube into a Collection Tube.
    • Load the lysate into the upper reservoir.
    • Centrifuge 1 min at 8,000 × g.
    • Discard the flow-through and Collection Tube.

  6. Inhibitor Removal

    • Insert Filter Tube into a new Collection Tube.
    • Add 500 μl Inhibitor Removal Buffer.
    • Centrifuge 1 min at 8,000 × g.
    • Discard the flow-through and Collection Tube.

  7. Wash Step 1

    • Add 500 μl Wash Buffer.
    • Centrifuge 1 min at 8,000 × g.
    • Discard the flow-through and Collection Tube.

  8. Wash Step 2

    • Repeat Step 7 with a new Collection Tube.

  9. Dry Spin

    • Centrifuge the column for 10 sec at full speed to remove residual buffer.
    • Discard the Collection Tube.

  10. Elution

    • Insert Filter Tube into a clean, sterile 1.5 ml microcentrifuge tube.
    • Add 200 μl pre-warmed Elution Buffer.
    • Centrifuge 1 min at 8,000 × g.

Storage:

Eluted DNA can be used immediately or stored at:

  • +2°C to +8°C (short-term; up to 3–7 days recommended)
  • –15°C to –25°C (long-term; up to 12 months)

⚠️ Disclaimer:

This protocol is a generalized example based on standard commercial DNA extraction kits. It has been anonymized and adapted for portfolio use only.