Bio-Rad Sequi-Gen GT Sequencing Cell - Manual (Page 26)

Condition Probable Causes Solutions/Preventions
5.2 DNA Sequencing Artifacts
Electrophoretic artifacts are described below. A DNA sequencing artifact may be defined
as any non-ideal graphic pattern on the X-ray film that reduces your confidence in reading, or
interpreting, a sequence from that film. There are three types of DNA sequencing artifacts:
• Template-dependent artifacts
• Electrophoretic artifacts
• Autoradiographic (or data acquisition) artifacts
Template-specific artifacts are caused by biological or chemical phenomena, and relate
to issues beyond the scope of this manual. Each sequencing method has its own set of potential
sequence-specific artifacts. Section 7.5 contains references that discuss sequence-specific
artifacts.
The basic premise for reading a DNA sequence is that each band on the film exists in a
vertical register that corresponds to one base in the sequence. Non-ideal patterns, caused by
problems in the three categories above, may interfere with the accurate determination of
DNA sequences. The following is a guideline for description and analysis of artifacts in DNA
sequencing gels, especially electrophoretic ones.
Electrophoretic Artifacts
There are many sources of electrophoretic artifacts. To simplify the task of defining an
artifact, we use a systematic description of electrophoretic artifacts, dividing all of the
possible patterns into three hierarchical sub-categories:
• Lane-local artifacts
• Set (template)-local artifacts
Upper buffer level drops too
fast during run
Sparks at the top of the gel
Sparks in lower chamber
Well-forming loading wells
deform when comb pulled out
Unexpected power conditions
Gel sticks to both plates when
opening sandwich
• Normal consequence of IPC
plastic bowing slightly as it
heats up
• Spacers leaking out the sides of
the gel
• Buffer leaks down between gel
and spacers
• Bond failure. Chamber leaking.
Sparks or burn marks in
adhesive.
• Upper buffer level dropped
below minimum level
• Lower buffer level too low or
too high
• Comb inserted too far
• Gel polymerized too long,
dried out
• Comb pulled out too quickly
• Gel not polymerized
• Buffers made incorrectly
• Gel hydrolyzed, more
conductive
• Gel too hot or cold
• Neither plate siliconized
• Both plates siliconized
• Plates unclean
• Outer plate pried off too quickly
• Refill upper buffer chamber
• Caution. Monitor run. Refill
upper buffer chamber.
• Polymerization problem
• Stop the run. Electrical hazard.
IPC needs replacement.
• Refill upper buffer chamber
• 350 ml is minimum, 500 ml is
maximum
• Insert comb minimum distance
• Rinse comb/gel with buffer
before pulling out comb
• Pull comb out slowly
• Refer to Bulletin 1156
• Check buffers
• Remake gel, run gel
cooler
• Run gel at 50 °C
• Siliconize outer plate according
to Section 3.1
• Separate plates slowly
23