( clone ALH 3000 to provide an automated ...)Automation of the Eppendorf Perfectprep BAC 96 DNA Kit on the Caliper Life Sciences Sciclone ALH 3000

Materials and Methods

Bacterial Culture Growth

1.5 mL of 2x YT growth medium was distributed into each well of a 96-well plate then inoculated with BAC clones from CITB Human BAC library plate #707. Four plates from the same quadrant were grown as replicates. The culture plates were incubated at 37 C for 24 hours while shaking at 325 rpm. The bacterial cells were harvested by centrifugation for 10 minutes at 1,900 x g.

Automated Workstation

- Sciclone Advanced Liquid Handler ALH 3000, (CaliperLS) with a High Volume Head (volumes up to 200 μL).
- Deck-mounted self-centering Microplate Shaker (CaliperLS) facilitates the resuspension of the pellet, mixing of the lysate and neutralization solution.
- Positive Pressure Filtration Accessory (CaliperLS) performs the filtration to clear the bacterial lysate, trap the BAC DNA and elute the BAC DNA.
- Gripper (CaliperLS) removes Filter Plate A, & transfer the BAC Filter Plate to the collection plate.
- Wide Bore Tips, 200 μL (Axygen, P/N ZT-205-WB-L-R) are used for the resuspension, lysis, neutralization and the transfer to Filter Plate A.
- Automation Certified Pipette Tips, 200 μL (P/N 56362) are used for pipetting of trapping, wash and elution reagents.

Automated Sample Processing

Sample processing is performed in quadruplicate using the Perfectprep BAC 96 kit on the Sciclone ALH 3000. Initially the reagents, sample plate, filtration plates and pipette tips are placed on the deck as shown on the Layout in the Sciclone software. As the automated sample processing starts, Solution 1 is added to the sample plate (located on the microplate shaker) using wide bore tip
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