Section III: Sequencing Troubleshooting Quick Reference Table
Sequence Analysis Troubleshooting Guide
31
• Poor Resolution
Check the accuracy
of base calling with
the pUC18 Control
Template.
Check pUC18 Control template—if
this is okay then the DNA template
and/or primer is at fault
or
If the pUC18 Control Template does
not meet the specifications
Poor Cycling
Bad Formamide
Problem in post reaction
cleanup of samples
Incorrect mineral oil
Capillaries
Gel
Use SLS solution to resuspend samples
Check Thermal Cycler
Problem with Ethanol precipitation
Check that glycogen was added
Centrifugation at 4
°
C
pH of Sodium Acetate
Concentration of EDTA
Check if mineral oil was added to
samples in plate
Check the life of the capillaries
Check life of gel
• Capillaries
Resolution is bad
Less than 6 peaks
per minute
Spurious peaks
Late start of data
even though current
is normal
The capillary has performed
more than 100 runs
The capillary coating has been
damaged
The tips were not kept moist
The array was not stored
correctly
Some type of chemical was
used to load the sample that
destroyed the coating (DMSO
etc.)
A sample plate was set up but
no corresponding buffer plate
was loaded
Purchase a new array
One or more of the
four dye traces are
>6000 counts in the
beginning of the run
Dirt on the window
Remove the capillary array at the
manifold end and clean capillaries. Use
sterile water or absolute ethanol.
DO
NOT USE METHANOL
. Clean in
one direction only
Manifold area dirty
Clean Manifold area
PROBLEM
RAW DATA
SIGNAL /
CURRENT
PROFILE
POTENTIAL SOURCE OF
PROBLEM
POSSIBLE SOLUTIONS
