Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide
5
Validation of the DTCS kit components
After determining that the CEQ System hardware, gel and capillary are functioning
properly, prepare sequencing reactions with the pUC18 control template and the -47
sequencing primer provided in the DTCS kit. This test is designed to try to
discriminate if the chemistry problems are due to the Beckman Coulter, Inc. provided
reagents or customer supplied reagents. Results that pass the system specification of
98% accuracy at 700 bases (using the LFR-1) confirm the quality of the Beckman
Coulter, Inc. reagents and the customer supplied materials except for primer and
template. If the test does not pass the 98% accuracy at 700 bases (with good raw data
signal and normal current profiles) perform the test again using a new DTCS kit
(preferably of a different lot number). Failure to pass the 98% accuracy with a second
kit would indicate that customer supplied reagents or the post sequencing reaction
cleanup method are suspect. If the cause of the problem is customer supplied reagents
proceed to the next section.
Suspect Customer Supplied Reagents
The customer uses several reagents that may have an impact on the raw data signal and
the current profile. These consist mainly of the chemicals used for the ethanol
precipitation of the finished sequencing reactions such as sodium acetate, EDTA and
ethanol. In some cases with older lots of DTCS kits customer supplied formamide may
also be an issue. The usual symptoms for poor quality sodium acetate, EDTA, ethanol
or formamide are low raw data signal and occasionally erratic current profiles. Using
sodium acetate and EDTA solutions supplied by Sigma as well as the Sample Loading
Solution (SLS) supplied in the DTCS kits will help eliminate problems caused by poor
quality reagents. If one of these reagents is suspect, replace each reagent in a
systematic approach (one at a time) to identify the faulty reagent. If the control
template passes the system specification of 98% accuracy at 700 bases (using the
LFR-1 method) and the Raw Data Signal and Current profiles look good, but the
customers templates do not yield acceptable results proceed to the next section.
Identification of Customer Supplied Template or Primer
Problems
After demonstrating that pUC18 control template and the -47 primer function
correctly, customer DNA templates should be sequenced. If possible, reagents should
be tested individually. This will allow the most efficient identification of problem
reagents. For example, if the customer is using a DNA template that can accommodate
the -47 sequencing primer, then side by side comparisons of sequencing reactions
performed with customer provided primer and the Beckman Coulter, Inc. provided
primer can be conducted.
Using the previously described 3-step process, a CEQ System user should be able to
identify and correct many of the most common problems found in CE based DNA
sequencing. Listed below are several DNA sequencing chemistry problem areas with
their corresponding symptoms and possible corrective actions.
