Section II: Troubleshooting a CEQ System Problem
26
CEQ™ 8000 Series Genetic Analysis System
3.
Purify the PCR products prior to using them in DTCS reactions.
4.
Use a primer that binds to a site internal to the primers used for PCR (nested
primer).
5.
Select annealing temperatures that limit the annealing of mismatched primers.
6.
Unambiguous sequence regions should be chosen for selecting the priming sites.
High Baseline Levels
High baseline levels can lead to erroneous base calling and short read lengths. Optimal
performance of the CEQ System is achieved when the baselines for the 4 channels
(blue, green, black and red) are below 6,000 cnts. Shown in Figure 19 are two
examples of “dirty capillaries”. The example on the left may yield acceptable quality
data. The example on the right shows an extreme case of a dirty capillary.
Corrective Action
1.
Remove the capillary array at the manifold end and clean the capillary window.
Use sterile water.
DO NOT USE METHANOL
. Clean in one direction only.
2.
Use “Direct Control” from the “Run” application to purge the manifold and to
fill the capillaries with new gel and then clean the capillaries (see step 1 above).
Figure 19: High Baselines
3 traces >6,000cnts
Blue >35,000cnts
Black >18,000cnts
Green >6,000cnts
