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

source plates, positions C2 and D2 respectively, were PCR plates. To avoid air bubbles being trapped in the bottom of the "V" shape of PCR wells, when dispensing the 10 μL of Denature buffer, we dispensed 5 μL of the volume very close to the bottom of the well, and then moved up 1 mm and dispensed the remaining volume. The pUC19 source plate was manually pre-loaded with 20 μL of DNA template (2 μg/mL) per well, so the final per well concentration is 1 ng of DNA.

Reservoir: The type of reservoir for holding Denature buffer simply needs to be sufficient to hold the required volume, and be made of a chemically compatible material.

Pipetting technique: The liquid classes used for this work are device dependent. For the Low Volume Head, aspiration and dispense speeds of 0.5 μL/sec were used with a 2 μL pre-sample air gap. For the Z-8, eight independent channel pipetting device, the liquid class settings included no pre or post air-gaps, aspiration and dispense speeds of 2000, Breakoff and Start Speeds of 500, and a backlash setting of 0 as shown in Figure 2. Aspiration and dispense speeds of 3 μL/sec, with a pre-sample air gap of 5 μL, were used for the mixing step in the DYEnamic kit.

The mean DNA template yield per-well was >3.0 μg and the %CV was <4.2 as shown in Figure 3. The smallest volume transfer was 0.5 μL. Results from the sequencing of the TempliPhi reaction are shown in Figure 4.

Discussion:

The results of this feasibility study show that liquid handling capabilities of the Sciclone ALH 3000 are sufficient to produce results that are consistent with the manual results expected for the TempliPhi and DYEnamic kits, which yield ~3.2 g of DNA per well, and readlengths of >600 bp. The use of the same Sciclone ALH 3000 configuration for both of these kits was done with furthe
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