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Automation of the Eppendorf Perfectprep BAC 96 DNA Kit on the Caliper Life Sciences Sciclone ALH 3000

s, and a combination of trituration and shaking is performed to resuspend the bacterial pellet. Next, wide bore tips are used to add Solution 2 to lyse the bacteria. Again the microplate shaker is activated to assure a homogenous solution. Wide bore tips are then used to transfer the neutralization solution, and a quick mix by trituration is done. The same wide bore tips are used to transfer the lysed sample to Filter Plate A.

Positive pressure is applied to clear the lysate of cellular debris and to transfer the samples to the BAC Filter Plate. A Gripper removes Filter Plate A. Trapping Buffer is added using standard pipette tips to the lysate in the BAC Filter Plate followed by applying positive pressure to trap the DNA on the membrane. The BAC DNA is further purified by filtering Diluted Wash Buffer through the BAC Filter Plate using positive pressure. Finally, the gripper is used to move the BAC Filter Plate on top of the collection plate and the BAC DNA was eluted using Elution Buffer using positive pressure.

Results

Agarose Gel Analysis

96 DNA samples are isolated in quadruplicate from quadrant A1 of BAC clones from CITB Human BAC library plate #707 using the Eppendorf Perfectprep 96 kit. A volume of 10 μL from 24 samples were run on a 1% agarose gel containing ethidium bromide (Figure 2).

Figure 2: BAC DNA isolated with the Eppendorf BAC 96 kit on the CaliperLS Sciclone ALH 3000. This shows 24 of the eluates. M: 300 ng of λHindIII.

DNA Sequencing Analysis

DNA sequencing is performed using conditions determined to be optimal for the Eppendorf Perfectprep BAC 96 kit. A volume of 10 μL of purified template DNA were used in all 384 sequencing reactions. The following sequencing reaction setup (Table 1) and cycling conditions (Table 2) were used:

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