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Yale University
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DNA Sequencing >
"No Analyzed Data"
Frequent reasons for a DNA sequencing
reaction
resulting in "No analyzed data"
"No analyzed data" results when there is an insufficient level of
fluorescent termination products for the computer software to call
bases.
- Poor quality template
DNA. Qiagen ion exchange resin (tip-20, tip-100, etc.) or
Qiawells seem to generate the best quality plasmid template DNA
for the widest number of researchers. Template DNA must be free of
residual ethanol and salt. Please refer to our sheet on plasmid
template preparation for more information.
- Insufficient amount of
template DNA. For double-stranded plasmids, 250-300 ng of
template DNA are required per reaction. We rely on the
investigator's quantitation of template DNA to set up the
reactions. In general, the more accurate the measurement of
template concentration, the better the quality of data that will
be returned (Too much DNA can be almost as bad as too
little).
- Insufficient primer
concentration.
We require primers at 4 µM
concentration which for a 20mer is equivalent to a about 27
ng/µl.
- Primer and/or template was not
added to the reaction. Occasionally, either the primer or
template is accidentally left out of a reaction. We try to
immediately identify and repeat such reactions.
- The tm of the primer is
<< 50 oC.
The annealing temperature in our cycle
sequencing reactions is 50 oC, therefore, all primers should have
a melting temperature Ñ 52 oC. Melting temperature of
primers should be determined by a program (Oligo or Primerselect)
which uses the nearest neighbor summation of thermodynamic
parameters to calculate an accurate tm. We recommend that all
sequencing primers be between 20 and 30 nucleotides in
length.
- The template does not contain
a sequence complementary to the primer. For example, the
pgem series of plasmid vectors do not contain a T3 primer site,
but instead contain an SP6 site.
- For PCR products,
occasionally one of the primers used to generate the PCR product
does not work in fluorescent cycle sequencing. It is likely that
such a primer, although sufficiently competent in the exponential
PCR process, is very inefficient in the linear amplification of cycle
sequencing.
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