Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide
19
Low Raw Data Signal Caused by Poor Quality Mineral Oil
The mineral oil supplied in the DTCS kit is high quality oil containing no detectable
nuclease activity. The use of other lower quality mineral oils can lead to sample
degradation and hence low signal as shown below. The red and black dyes are
particularly susceptible to this problem.
Corrective actions
1.
Repeat the sequencing reactions and use the mineral oil supplied in the Beckman
Coulter, Inc. CEQ DTCS kits.
2.
Repeat the sequencing reactions and use molecular biology grade mineral oil
from Sigma Chemical Company (P/N M-5904)
Low Raw Data Signal Caused by Thermal Cycling Problems
The CEQ DTCS chemistry uses thermal cycling to produce a linear amplification
process (called cycle sequencing) to generate sufficient fluorescent signal to generate
an accurate sequencing base call. The Thermal Cycler program and performance are
critical to achieve optimal results. There are several possible problems that are
associated with the thermal cycling process. These include:
•
Using the wrong thermal cycler or a poorly performing Thermal Cycler
•
Using the wrong ramping setting on a thermal cycler
•
Using an incorrect annealing temperature for the primer being used for sequencing
•
Using a tube or plate that does not properly fit the thermal cycler
•
Using a thermal cycler without a heated lid
•
Loose fitting strip caps, crimped sealing film or sealing mats
Figure 13: Sample degradation resulting from low quality mineral oils
