Section II: Troubleshooting a CEQ System Problem
16
CEQ™ 8000 Series Genetic Analysis System
Corrective Actions
1.
Add the correct amount of the primer to the reaction. This will require
quantitation of the primer by spectrophotometry. The recommended amount of
primer is at least 40-fold molar excess in relation to the template. For sequencing
of most template DNAs, 3–10 pmoles of primer is sufficient for a successful
sequencing reaction.
2.
Make sure that the primer preparation is pure. Using reputable oligonucleotide
vendors is your best way to get high quality primers. If possible have the primers
purified by TSP or HPLC.
3.
Always design the sequencing primers for use in Cycle sequencing reactions.
This means designing primers with high T
m
’s and no possibility of forming
secondary structures or primer dimers. There are many DNA analysis programs
available for checking primer design.
4.
Make sure that the primer is resuspended in a solution that has no EDTA or
DEPC present. Molecular biology grade water or 10mM Tris pH 8.0 are good
resuspension solutions.
5.
Increase the number of cycles in the thermal cycling program from 30 to 40 or
50.
