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Oil-Free PCR Using the Hot Top Assembly

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Duplicate cycle sequencing reactions were set up and performed under identical cycling parameters on a RoboCycler 96 temperature cycler. The first set of reactions was performed with the standard paddle and an oil overlay. The standard paddle was then removed and replaced with the Hot Top Assembly. The second set of reactions was prepared with the oil overlay omitted and cycled using the Hot Top Assembly. After cycling, equal volumes of the sequencing products were electrophoresed on a sequencing gel using the CastAway system.6 The gel was fixed, rinsed with water, dried and exposed to film. When either the standard paddle or the Hot Top Assembly was used, equivalent cycle sequencing results were obtained (figure 3). Similar results were observed when the experiment was performed using a RoboCycler 40 temperature cycler (data not shown).

Conclusions

By heating PCR tubes from both the top and sides, the Hot Top Assembly for RoboCycler temperature cyclers dramatically reduces the amount of condensation formed during cycling and eliminates the need to add oil or wax overlays to reaction tubes. The Hot Top Assembly makes postcycling manipulations, such as gel loading, significantly faster and easier, especially when many samples are analyzed. Our results demonstrate that the addition of the Hot Top Assembly to RoboCycler units did not compromise the quality of PCR or cycle sequencing results when compared to those obtained using a standard paddle and an oil overlay. By installing the new software upgrade PC card, which comes with the Hot Top Assembly for the RoboCycler 96 unit, any RoboCycler 96 temperature cycler can be retrofitted to become an oil-free PCR machine. Hot Top Assemblies are now available for any RoboCycler 96 or RoboCycler 40 temperature cycler,
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