DNA Engine Operations Manual
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Problems Related to Protocols
Following is a general description of some common problems related to the protocols
and reaction components in sequencing and amplification applications. For a more
detailed discussion of protocols and reactions, see Current Protocols in Molecular
Biology (F. Ausubel et al., eds., John Wiley & Sons), specifically chapters 7 (DNA
sequencing), 14 (in-situ hybridization and immunology), and 15 (polymerase chain
reaction).
Error Message Cause and Result Action
Reaction is working, but
broad low-molecular
weight band is seen
in gels.
“Primer-dimer” material often pro-
duces a broad band in the <100 bp
region of gels.
If obtaining appropriate reaction
product/s, no need to change
anything.
Minimize “primer-dimer” production
by designing primers with no 3'
self-complementary.
Reoptimize magnesium concentra-
tion and annealing temperature to
maximize desired product and
minimize “primer-dimers”.
Reaction working, but
unexpected extra prod-
ucts or smear is seen.
Nonspecific hybridization occurring
during setup.
Program a hot start into the
protocol.
Reaction component concentration
too high or too low.
Check concentrations of compo-
nents. May need to reoptimize
magnesium concentration.
Annealing temperature too low. Reoptimize annealing temperature.
Protocol contains a wrong value. Use List utility to check protocol’s
temperature control method, tem-
peratures, and times.
Template not of sufficient purity. Check extraction and purification
protocols. Add additional purifica-
tion steps if necessary.
Multiple templates or host DNA in
sequencing reactions.
Check nucleic acid preparations
by gel electrophoresis.
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