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Scalable Automation of the TempliPhi DNA Sequencing Template Amplification and DYEnamic ET Terminator Sequencing Kits on the Sciclone ALH 3000

or TempliPhi Premix, which contains the active enzyme. These conditions were the primary concerns driving the decisions for selection of the automation presented in this note.

In addition, we addressed throughput scalability with a method that allows the user to select whether the Premix source will be a microcentrifuge tube or a column reservoir. Using IF/THEN type method control, at the beginning of the method a user can specify the Premix source container. For low throughput situations, the user may choose to use only a single microcentrifuge tube, while for higher throughput applications the user can specify a column reservoir with a higher capacity. Even higher capacity reservoirs would be simple to include in the method, however, we choose to do all our work in a single temperature controlled reservoir which has both tube and column options available. The enzyme in the Premix is the most costly component of the TempliPhi kit, and temperature control of the Premix is necessary to maintain enzyme activity.

Timing comparison results are presented for 96-well plate format for both tube and reservoir sources of Premix.

Materials and Methods

Instrumentation

Liquid Handler (Sciclone ALH 3000 with software version 3.1.14, firmware version 1.14 [CaliperLS]). The Sciclone was equipped with the following:

- Low Volume Head (P/N 103453/0)
- 96 Mandrel Array (P/N 103969/1)
- Press Station (P/N 104826/0)
- Z-8 Pipettor (P/N 102425/C)
- Temperature-Controlled Locator (P/N 103412)
- Temperature-Controlled Locator Block (P/N 103481), includes 3 column reservoirs and holds 12 microcentrifuge tubes.
Thermocycler (MJ Research PTC-200)
Capillary Sequencer (Amersham BioSciences MegaBACE 1000)

Materials:'"/>

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