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

e cycle-sequencing extension products were purified by precipitation with EDTA and 95% high purity ethanol followed by one wash with 70% high purity ethanol. The purified samples were resuspended in 8 μL of 0.1x TE and loaded on an ABI PRISM 3700 DNA Analyzer using the default run modules for POP-5 polymer and an injection time of 90 seconds. Data analysis was performed using Phred base-calling software (CodonCode Corporation, version 0.020425.c) and passing scores were determined to be those samples with greater than 100 Phred Q20 bases, shown in Figure 3. The sequencing results are shown in Table 3.

Discussion

Initial experiments indicate that the Eppendorf Perfectprep BAC 96 kit can be readily automated on the Sciclone ALH 3000. Results are reproducible from plate to plate and high quality DNA is obtained resulting in average sequencing read lengths > 590 bp with 96% pass rates. The versatile Sciclone ALH 3000, using an innovative gripping module, a leading-edge Positive Pressure Filtration system, an on-board shaker module, and a 96-tip head, proves to be an efficient system to obtain 1-16 plates of high-quality BAC DNA in a day. For higher throughput laboratories, a Twister II Plate Handler (CaliperLS) can be incorporated into the system, and scheduled though the CLARA Software (CaliperLS) to provide a higher capacity and throughput system based on the needs. This combination of the easy-to-use Eppendorf Perfectprep BAC 96 kit and the Caliper Life Sciences Sciclone ALH 3000 provide sufficient quantities of BAC DNA suitable for all downstream applications necessary for assembly of large-genome constructs.


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