( tiple fluorescent tags are used for cyc...)Automated Fluorescent-Tag Cycle Sequencing

tiple fluorescent tags are used for cycle sequencing, uniform signal strength among the different tags is especially important for scan and base-calling accuracy. A temperature cycler block that does not produce equal cycling conditions in each well may not incorporate the dideoxy nucleotide reaction terminators at a uniform rate between sample wells. Incorporation of fluorescent tags that is not uniform results in uneven band intensities, which are difficult to scan. Thus, the sequence of the resulting cycle sequencing product cannot be determined accurately.

figure 1

Stratagenes line of RoboCycler temperature cyclers have unmatched well-to-well temperature uniformity during PCR amplifications (figure 1). The addition of the new Hot Top Assembly, which is available for both gradient and nongradient RoboCycler 40 and 96 models, eliminates the need for an oil or wax overlay on reactions. The Hot Top Assembly prevents sample evaporation even for the small reaction volumes that are typically used in cycle sequencing reactions. We determined the accuracy with which the RoboCycler Gradient 96 temperature cycler with a Hot Top Assembly can perform fluorescent-dye cycle sequencing reactions.

Comparison of Temperature Cyclers

Cycle sequencing experiments were performed using both Stratagenes RoboCycler Gradient 96 temperature cycler with Hot Top Assembly and the GeneAmp PCR System 9600 cycler. The ABI PRISM dye terminator cycle sequencing ready reaction kit was used to sequence the kits control plasmid, the pGEM -3Zf(+) vector, using the (-21) M13 forward primer. Both experiments were performed according to the manual supplied with the ABI PRISM sequencing kit. Following the completion of cycle sequencing, the 20-l reaction volume
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