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- The Ion Sphere Particles (ISPs), included in the template
preparation kit, should then be vortexed at maximum speed
for 1 min and immediately 100 μL should be added to the
900 μL of amplification solution. - The filter assembly should then be prepared and installed.
- By using a pipette, add the 1 mL prepared solution and 1.5 mL
of OneTouch oil to the reaction filter. - The filter should be inverted and inserted into the OneTouch.
- An assisted run should be selected from the dropdown menu
and after following each prompt, the run should be started. - The OneTouch should be cleaned according to the instruction
after each use. - Both of the recovery tubes should be removed immediately
after the last centrifugation step in the OneTouch and all but
50 μL of recovery solution should be removed using a pipette. - Care should be taken not to disturb the pellet and the tem-
plate-positive ISPs should be resuspended in the remaining
recovery solution. - The ISPs should then be washed in 1 mL of Ion OneTouch
wash solution. - At this stage, ISPs could be stored for up to 3 days at 4 °C.
- Just before sequencing, ISPs should be centrifuged for 2.5 min
at 15,500 × g, and all but 100 μL of supernatant removed. - The pellet should then be resuspended by vortexing.
- Prepare the Ion OneTouch Enrichment system (Fig. 3b).
- Prepare the melt off solution by combining the components in
the following order:
Nuclease-free water (277 μL), 1 M sodium hydroxide
(40 μL), and 10% tween 20 detergent in nuclease-free water
(3 μL). Total volume is now 320 μL. - Fill the 8-well strip with template-positive ion sphere particles,
magnetic beads, 300 μL of wash solution, and 300 μL of melt
off solution in order leaving the sixth and eighth well empty
and then perform the run. - At the end of the run, remove the PCR tube containing the
enriched ion sphere particles, which should be more than
200 μL. - Wash the ion sphere particles and proceed for sequencing (see
Note 4).
3.2.1 Recovery
of Template-Positive Ion
Sphere Particles (ISP)
3.2.2 Enrichment
of Template-Positive Ion
Sphere Particles (ISP)
Suja Pillai et al.