Section II: Troubleshooting a CEQ System Problem
18
CEQ™ 8000 Series Genetic Analysis System
Low Raw Data Signal Due to Insufficient Sample Injection
Poor injection of DNA sequencing fragments onto the CEQ capillaries will lead to low
Raw Data signal. Since the CEQ uses electrokinetic injection it is highly sensitive to
excess salts in the loading solution. The excess salts compete with the DNA
sequencing fragments during injection and result in lower loading of the fluorescently
labeled DNA molecules. The sources of the excess salts are improperly purified
sequencing reactions and decomposed formamide.
Corrective actions
1.
Follow the ethanol precipitation procedure in the CEQ Dye Terminator Cycle
Sequencing protocol (Beckman Coulter, Inc. P/N 608019).
2.
If using spin column purification methods make sure that the column materials
do not contain salts (check with the spin column manufacturer for details for
using their products with capillary sequencers).
3.
Use Beckman Coulter, Inc. supplied SLS for sample resuspension.
Figure 12: Sequencing reaction
Sequencing Reaction with excess salts
Sequencing Reaction with no impurities
