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

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96-well PCR microplates (MJ Research Hard-Shell Thin Wall P/N HSP-9601)
Tempo-100 Pipet Tips (P/N 66670)
Tempo 25 μL disposable tips (P/N 104035)
pUC19 DNA control template (United States Biochemicals P/N 76632)
TempliPhi DNA Sequencing Template Amplification Kit (Amersham BioSciences P/N 25-6400-01)
DYEnamic ET Terminator Sequencing Kit (Amersham BioSciences P/N US81095)
PicoGreen dsDNA Quantitation Kit (Molecular Probes P/N P-7589)

Deck Layout: For the TempliPhi Kit, the deck layout is shown in Figure 1. Position A1 is a tipbox, A2 is tip disposal, B2 is the Denature buffer reservoir, C2 is the TempliPhi reaction plate, D2 is the pUC19 DNA source plate, A4 is the temperature-controlled locator containing the Premix in either tube or column reservoir format, and C5 is the Press Station (used for attaching 96 disposable tips to the 96 Mandrel Array). For the DYEnamic Kit, the same layout is used, except positions A2 and B2 are left empty, while the temperature-controlled reservoir at A4 is used to hold the Premix solution and the product plate from the TempliPhi reaction is now used as the source plate in position D2. The Premix contains enzyme, dNTP's, and ddNTP's and is supplied in the DYEnamic Kit. Prior to the run, this was manually mixed with 5 μM primer (M13 Forward) and water.

All Sciclone ALH 3000 movements were performed at 100% travel speed except when entering and exiting liquids. Liquid entry/exit was typically done at an approach speed of 20% and a retraction speed of 5%.

Pipette Tips: Automation Certified Tempo-100 tips were used for all Z- 8 pipetting. For Mandrel Array pipetting, Tempo 25 μL tips were used (P/N 104035).

Plates: For the TempliPhi reaction, the reaction plate and DNA
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