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Time Considerations
When starting with up to six samples of
precipitated RNA, the protocol, including second-round amplification with a subset of all
primer combinations, can be performed within
two days, including an overnight ligation step.
The remaining set of second-round amplifications can be done at a rate of four to six 96-well
plates per day and person. Alternatively, employment of a robotic pipetting station might
be considered. Choosing the DBE variant, two
membranes per day per DBE machine can be
prepared, each providing space for 30 reactions. It should be noted that buffer capacity
allows for using each DBE gel twice, provided
that the second run starts immediately after the
first run without idle electrophoresis in between. One person can then operate three to
four machines per day and produce 6 to 8
membranes. In such a medium-scale setup, gels
are prepared in the evening, and, with edges
carefully wrapped in plastic wrap with some
wetted pieces of paper towel enclosed, allowed
to polymerize overnight. In the morning, gels
are mounted and electrophoresis is started.
During electrophoresis, the membranes of the
day before are developed, the glass plates of the
previous runs cleaned, and the gels for the next
day are prepared.
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Contributed by Achim Fischer
F. Hoffmann-La Roche AG
Basel, Switzerland
Discovery of
Differentially
Expressed Genes
25B.4.17
Current Protocols in Molecular Biology
Supplement 56