Pipelines to do MinION sequencing of Zika virus

Post-PCR clean-up and amplicon quantification

Clean-up

  1. Label two sets of DNA Lo-Bind tubes (one set for bead clean-up and the other for eluate with cleaned up amplicons)
  2. Allow AMPure XP beads to come up to room temperature, and homogenize by vortexing.
  3. Add 72 uL of AMPure XP beads to each tube in one set of the Lo-Bind tubes. this is a 1.8x bead clean-up
  4. Pipette 40 uL of PCR product into correspond tube with beads.
  5. Incubate beads and PCR products at room temperature on a hula mixer for 5 minutes. if no access to a hula mixer, you can flick mix gently throughout the incubation
  6. Place tubes on magnetic rack and incubate until the solution is clear.
  7. Discard the supernatant being careful to not disturb pellet.
  8. Add 200 uL of 80% EtOH to each tube to wash pellet, incubate 30 seconds, then discard EtOH wash.
  9. Repeat previous wash again, pipetting off as much EtOH as possible.
  10. Spin tubes down, replace on magnetic rack, and pipette off any additional EtOH. Leave tubes open to dry.
  11. Allow pellets to dry (roughly 5 minutes). The pellet should appear matte but not dry to the point where cracks form.
  12. When pellet is dry add 31 uL of nuclease-free water to each tube, remove from rack and flick gently to resuspend beads. if storing amplicons for longer periods of time, qiagen Elution Buffer can be used instead of nuclease-free water.
  13. Once pellets have been resuspended incubate at room temperature on a hula mixer for 5 minutes.
  14. Replace tubes on magnetic rack and incubate until solution fully clears.
  15. Carefully pipette off 31 uL of supernatant without disturbing beads and place into new Lo-Bind tubes.

Quantification

You should use the Qubit High Sensitivity dsDNA kit

  1. Make up Qubit Mastermix in a falcon tube :
  • 199 uL Buffer per sample
  • 1 uL dye per sample
  1. Add 190 uL mastermix + 10 uL standard 1 into one Qubit tube.
  2. Add 190 uL mastermix + 10 uL standard 2 into another Qubit tube.
  3. Add 199 uL mastermix + 1 uL cleaned-up PCR products into Qubit tube for each pool.
  4. Vortex, spin down and wait 2 minutes.
  5. Quantify the concentration of dsDNA in the sample.
  • Negative extraction controls usually have concentrations around 3 or 4 ng/uL.
  • Negative PCR controls should have concentrations < 1 ng/uL.
  • Properly amplified samples should have between 5 and 100 ng/uL.