Troubleshooting chip images using the Chip View
The following table summarizes some of the more common problems that can affect imaging of Digital PCR 20K Chips. In the examples shown below, the conditions triggered a data flag and were diagnosed using the Chip View of the AnalysisSuite™
Software.
Observation Possible Cause Action
• Excess PCR reaction was present on the Digital PCR 20K Chip after loading it with the Sample Loading Blade. • (Manual load only) The
Sample Loading Blade was drawn across the chip too quickly or at an angle shallower than 70-80°.
If possible, use the
AnalysisSuite™ Software to filter
the low quality data points, or discard the chip and run the sample again.
To prevent leaving excess PCR reaction on the Digital PCR 20K Chips:
• Make sure to install theTilt Base to the thermal cycler. You must thermal cycle the Digital PCR 20K Chips at an 11° angle.
• (Manual load only) Confirm that the heated block used to load Digital PCR 20K Chips is set to 40±1°C.
• (Manual load only) Make sure to wait 20 seconds after loading a Digital PCR 20K Chip before pre-wetting it with Immersion Fluid. • (Manual load only) Make
sure to draw the Sample Loading Blade across the chip slowly (>10 seconds) and at a 70-80° angle.
Observation Possible Cause Action
A bubble was present in the Sample Loading Blade when it was used to apply the PCR reaction to the Digital PCR 20K Chip.
If possible, use the
AnalysisSuite™ Software to filter
the low quality data points, or discard the chip and run the sample again.
When loading the Sample Loading Blades:
• If you are using a manual pipette, pipette to the first stop.
• Decrease your pipetting speed.
Troubleshooting chip images using the Chip View
Observation Possible Cause Action
• The Digital PCR 20K Chip leaked during thermal cycling or imaging.
• A large bubble was present in the chip (insufficient Immersion Fluid).
• Excess Immersion Fluid is present on the Chip Case Lid.
If present, remove excess Immersion Fluid from the chip lid and run the chip again.
If possible, use the
AnalysisSuite™ Software to filter
the low quality data points. To minimize leakage:
• Leave a small bubble when filling chips with Immersion Fluid.
• When covering chips with Immersion Fluid, do not get fluid on sides of the chip base.
• When sealing the chips, create a dome of sealant over the fill port.
• Cure the Chip Sealant under ultraviolet light for at least 30 seconds.
If you are sealing chips using a Chip Loader, make sure that you are using at least 20 lbs of pressure when applying the chip lid. If you are unsure that you are applying sufficient force, increase the duration of the press.
If you are sealing chips manually, also do the following to minimize leakage:
• Wear correctly fitted gloves to prevent the material from snagging during lid
application.
• Make sure that the Chip Case Lid is correctly aligned to the Chip Case.
• Firmly press all four corners when applying the Chip Case Lid.
• When pressing the Digital PCR 20K Chip, firmly hold the sides of the chip and wiggle it back and forth.
Chapter 5 Troubleshooting
Observation Possible Cause Action
Chip was not completely covered with Immersion Fluid
immediately after loading (while still on the chip loader or heated block)
Note: (QuantStudio® 3D
Instrument firmware version 1.1 or less) Yellow around the edges is a strong indication of
evaporation.
Add Immersion Fluid as soon as the loading blade is off of the chip.
Ensure that entire chip is covered with Immersion Fluid, even the edges.
Note: When adding Immersion Fluid, the fluid can overflow into the chip base, but do not allow it to spread onto the sides of the chip lid.
Immersion Fluid was not applied to the chip immediately after loading (evaporation of the PCR reaction).
Debris or other contaminants are present on the Chip Sealant during imaging.
No action required.
The AnalysisSuite™ Software can
compensate for small quantities of debris or other contaminants on the Digital PCR 20K Chip.
Troubleshooting chip images using the Chip View
Observation Possible Cause Action
Condensation may have been present on the Digital PCR 20K Chip during imaging.
IMPORTANT! Condensation may not be visible on the Digital PCR 20K Chip.
• If condensation is not visible – If possible, read the Digital PCR 20K Chip again after it has warmed to room temperature. Once
equilibrated, the data quality, flag, and separation of amplified versus non- amplified peaks should improve noticeably. • If condensation is visible –
Using a low-lint wipe, remove any condensation or Immersion Fluid from the surface of the chip by wiping in one direction. If
necessary, use a low-lint wipe sprayed with
isopropanol to remove any dried residue.
The Sample Loading Blade was not seated correctly within the loader arm.
Before pressing the loading button, confirm that the Sample Loading Blade is firmly seated and flush against the loader arm.
Chapter 5 Troubleshooting
Observation Possible Cause Action
Excess template was loaded onto
the chip. In the QuantStudio
® 3D
AnalysisSuite™ Software, check
the copies per reaction and dilute the sample accordingly so that it is within the digital range.
Troubleshooting chip images using the Chip View
Observation Possible Cause Action
Too much sample was left in
loading blade. Ensure that you are loading only14.5 µL of reaction mix into the loading blade.
Chapter 5 Troubleshooting