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8 / Re-run calibration measurements to ensure system stability
A recording is not a meaningful measurement until it has been calibrated with
a calibration recording that has been run under the same conditions (stretch,
pressure, voltage and baseline current). If the sample recording has not been
calibrated properly, the errors for size and concentration can be significant.
For size analysis only, measurements need to only be taken at one pressure.
In this case, run one calibration, and then run the sample under identical
conditions.
For size and concentration analysis, both the sample and calibration
measurements must be taken at a minimum of 2 (ideally 3) pressures. Current
and particle rate should be recorded for each run. The measurement plan for
multiple samples may look like this:
— Calibration 1 (P10, P6, P3), Current 131 nA
— Sample 1 (P10, P6, P3), Current 131 nA
— Sample 2 (P10, P6, P3), Current 136 nA
— Calibration 2 (P10, P6, P3) Current 135 nA
— Sample 3 (P10, P6, P3), Current 141 nA
— Sample 4 (P10, P6, P3) Current 143 nA
— Calibration 3 (P10, P6, P3) Current 144 nA
Additionally, nanopores are prone to stretching over time, so it is important to
use the correct nanopore size and recalibrate the pore often.
If the baseline current increases by more than 3%, this indicates an irreversible
change in nanopore size, and the pore must be re-calibrated.
If the baseline or particle rates drop by more than 5%, or the noise increases
past the acceptable range of 5–9 pA, this indicates a partial nanopore
blockage. Consult the troubleshooting guide and restore the system stability
before repeating the recording.
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