Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide
17
Low Raw Data Signal Due to “Bad Formamide”
Formamide is used to resuspend the DNA sequencing fragment prior to loading on to
the CEQ. The formamide solution must be prepared and stored properly to achieve
high quality sequencing data. If the formamide is not deionized and stored properly it
will decompose into ammonia and formic acid. The formic acid then destroys the
fluorescent dyes and produces low Raw Data signal. Shown in Figure 11 is a sample
that was resuspended in formamide that had not been deionized. Note that the resulting
base calling accuracy for this sample was less than 90% (>40 errors) at 500 bases.
Corrective actions
1.
Use the Beckman Coulter, Inc. supplied Sample Loading Solution (SLS).
2.
Do not freeze-thaw the SLS or formamide solutions. Store aliquots at –20°C in a
non-frost free freezer and use the aliquots only once.
Low Raw Data Signal Due to the Use of Water as the Sample Loading
Solution
We do
not
recommend using water to resuspend the DNA sequencing fragments prior
to loading on the CEQ. The Beckman Coulter, Inc. WellRED™ dyes are not stable in
pure water solutions and will yield Raw Data signals similar to that of “Bad
Formamide” (see prior section).
Corrective actions
1.
Use the Beckman Coulter, Inc. supplied Sample Loading Solution (SLS).
2.
Do not freeze-thaw the SLS or formamide solutions. Store aliquots at –20°C and
use the aliquots only once.
Figure 11: Sample resuspended in formamide that has not been deionized
