DNA Sample Processing Protocol

Workspace Setup

• Ensure proper workspace flow to avoid contamination.
  • Set up for right-handed students:
    • Tips and pipettors on the left.
    • Racks and samples in the middle.
    • Discard bucket on the right.
• Hand movement should not cross over samples unnecessarily.

Sample Preparation

• Following steps occur after students have added buffer to their DNAgenotek collection kits and mixed the solution by inversion.
• Samples are not labeled to preserve anonymity.

Transferring Spit Samples

• Use a P1000 micro-pipettor to transfer 500 microliters from each spit collection tube to a new microcentrifuge tube.
  • Draw solution slowly to avoid bubbles or contamination.
  • Close spit tube lid and cap the new sample tube.
  • Repeat for all samples.

Heat Block Incubation

• Heat block should be set to 50 degrees Celsius and turned on.
  • Indicator light will glow red when heating.
• Place samples in heat block for at least 90 minutes or overnight.

Adding Purifying Solution

• Tubes containing 20 microliters of DNAgenotek's proprietary purifying solution (PrepIT) provided.
• After incubation, transfer the entire volume to the tube with purifying solution.
  • Solution may become cloudy (normal).
• Mix the solution using a vortex:
  • Set vortex to Touch mode and maximum speed.
  • Press tube against black pad to initiate vibration.
  • Repeat for all samples.

Ice Incubation

• Place all samples on ice for 10 minutes.

Centrifugation

• Remove impurities by centrifugation.
  • Turn on centrifuge and open lid.
  • Remove metal lid inside by pulling the knob.
  • Label tubes with letters or numbers.
  • Load tubes with hinges facing outward, balancing volumes across the rotor.
  • Replace the metal lid securely.
  • Close centrifuge lid and set time (5 minutes) and speed (14.5 thousand RPM).
  • Press Start and monitor until maximum speed to ensure no violent vibrations.
  • If vibrations occur, press Stop immediately.

Post-Centrifugation

• Obtain four new tubes and label them.
• When spin completes, open lid and gently remove samples to avoid disturbing the pellet.
• Align tubes of the same sample for ease of transfer.
• White pellet at the bottom of the tube contains impurities; DNA is in the supernatant.

Transferring DNA

• Use P1000 to transfer 400 microliters of supernatant to a new tube.
  • Draw from the opposite side of the tube to avoid disturbing the pellet.
  • Repeat for all samples.