Section II: Troubleshooting a CEQ System Problem
Sequence Analysis Troubleshooting Guide
13
Shown, in Figure 8, is an example of a DNA sequencing sample where not enough
template DNA was added. In this example less than 25 fmoles of template was added
to the sequencing reaction. Notice the very low Raw Data signal throughout the run
and particularly the low Raw Data signal in the red, green and black traces. An easily
identifiable indicator of low template concentration and, in general, a poor sequencing
reaction is a high amount of unincorporated dye-terminators (note the large peaks at
around 40 minutes).
Corrective actions
1.
Add the correct amount of the template DNA to the reaction. This will require
quantitation of the template DNA by spectrophotometry (in the case of
commercial DNA preparations) or by estimation using agarose gel
electrophoresis and comparison to a know quantity of DNA.
2.
Alternatively, the user could try a dilution series with the same template starting
with an amount that is obviously too high and ending with an amount which is
much too low. This method assumes that the user knows the approximate amount
of template added to the reaction (this may be from previous work using similar
DNA preparation methods).
3.
Use the preheat treatment for highly supercoiled plasmids. Most commercial
DNA preparations yield highly supercoiled plasmids. The preheat treatment will
knick the supercoiled plasmid which yields much more efficient DNA
sequencing (linear molecules sequence better than supercoiled molecules).
Figure 8:
Low Template amount
Low signal
Unincorporated
Dye-Terminators
