Pipelines to do MinION sequencing of Zika virus
Source code
Latest commits

Post-PCR clean-up and amplicon quantification



  2. Allow AMPure XP beads to come up to room temperature, and homogenize thoroughly by vortexing.
  3. Add 40 uL (1x) or 72 (1.8x) uL of AMPure XP beads to each tube in one set of the Lo-Bind tubes depending on sequencing platform.
  4. Pipette 40 uL of PCR product into corresponding tube with beads.
  7. Discard the supernatant being careful to not disturb pellet.
  8. Add 200 uL of 80% EtOH to each tube to wash pellet (while being careful to not disturb pellet), incubate 30 seconds, then discard EtOH wash.
  11. Allow pellets to dry (roughly 5 minutes). The pellet should appear matte but not dry to the point where cracks form. DNA yield will drop if the pellet is left to dry too long.
  13. Once pellets have been resuspended, incubate at room temperature on a hula mixer for 5 minutes.


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
  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. If you have access to gel electrophoresis, you can run these negatives on a gel to check whether there is amplicon contamination or whether the quantifiable levels of dsDNA are simply represent a smear.
  • Negative PCR controls should have concentrations < 1 ng/uL. Ideally these are unquantifiable, but it is challenging to get concentrations this low except in completely ideal lab settings.