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Automated Fluorescent-Tag Cycle Sequencing

Application of the RoboCycler Gradient 96 temperature cycler

Douglas Drake
Stratagene Cloning Systems, Inc.

Stratagenes RoboCycler Gradient 96 temperature cycler*,# with Hot Top Assembly can be used to perform fluorescent-tag cycle sequencing using the ABI PRISM dye terminator cycle sequencing ready reaction kit. The new Hot Top Assembly eliminates the need for oil or wax sample overlays that hinder the loading of cycle sequencing reactions onto polyacrylamide gels. With unmatched well-to-well temperature accuracy, the RoboCycler Gradient 96 temperature cycler ensures the even and reproducible reaction conditions that are essential for achieving uniform fluorescent-tag signals and better read accuracy.

Cycle sequencing, using fluorescent dye-tagged terminators or primers, has become the standard protocol for use with automated sequencing devices.1 The fluorescent dye-labeled cycle sequencing products are loaded onto a vertical denaturing polyacrylamide gel and separated by gel electrophoresis. Automated gel reading systems contain one or more lasers that excite the fluorescent tag and sensors that detect fluorescent emissions within each lane. These systems can typically detect and distinguish different fluorescent tags on the basis of emission wavelength and quantitate the strength of fluorescent signals at each reaction termination point. By detecting the fluorescent-dye tags along the electrophoretic axis of the gel, the instrument can automatically determine the correct sequence of the reaction template with greater than 98% accuracy.2

PE Applied Biosystems have found that, for their ABI PRISM 377 and 373 DNA sequencers, poorly calibrated temperature cyclers can have a dramatic effect on the sensitivity, specificity and reproducibility of cycle sequencing reactions.3 When mul
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