Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide
9
Corrective actions to solve erratic current or current crashes
The occurrence of erratic current profiles indicates that there is a more severe DNA
template-overloading problem. In this case more drastic measures may need to be
taken. In the situation were erratic current is observed, simply rerunning the sample
and injecting for half the time will not solve the problem. Listed below are some of the
steps the should be made to eliminate the erratic current problem:
1.
If sufficient raw data signal is present, reducing the amount of template present
in the cycle sequencing reaction is an obvious first step. In this case, adding half
the amount of template may eliminate the current problem (i.e. if the original
template amount was 100 fmoles, use 50 fmoles).
2.
Use of the preheat treatment of the template DNA prior to the cycle sequencing
reaction is highly recommended for supercoiled plasmid samples and is required
to solve erratic current problems or current crashes. Once again, the preheat
treatment (see CEQ DTCS Chemistry, Beckman Coulter, Inc. P/N 718119) nicks
the template DNA to alleviate the supercoiling of the plasmid DNA and nicked
DNA does not “clog” the capillaries during injection. In many cases the preheat
treatment of plasmid templates at 96°C for 1 minute prior to adding the template
to the sequencing reaction can eliminate current irregularities (see Figure 6).
Figure 6:
Preheat treatment of plasmid templates
