Lynn Jordan, James Batchelor, and Ed Alderman
Applied Science and Technology Department, Caliper Life Sciences Inc., Hopkinton, MA 01748
Kelly M. Clark, Joseph A. Hensley and Robert P. Weisbein
Brinkmann An Eppendorf Company, Westbury, NY
Jennifer L. Halcome and George R. Halley
Eppendorf 5 Prime, Inc. Boulder, CO
To assemble large genome constructs, Bacterial Artificial Chromosomes (BACs) are necessary for fingerprinting mapping and BAC-end sequencing. It is critical to have an automated method to obtain high quality BAC DNA. We have completed feasibility studies integrating Eppendorfs Perfectprep BAC 96 reagent kit with Caliper Life Sciences Sciclone ALH 3000 to achieve high quality BAC in a timely fashion. A 96- channel pipetting head performs reagent additions using disposable tips. An on-deck shaker is used to resuspend the pelleted material, and is used for mixing in other steps. Positive pressure is used to perform all filtrations steps. Through the use of an on-deck gripper, plates can be relocated on the deck to provide a Hands-Off automated solution.
Purification of sufficient quantities of high quality BAC DNA, when using lower volumes of reagent and starting culture, has posed a challenge for researchers. Eppendorf has solved this problem with the development of the Perfectprep BAC 96 that is capable of processing 96 samples in a microplate format. Figure 1 shows an overview of the Prefectprep BAC 96 process. For many researchers, projects involve the processing of thousands of samples to acquire the downstream data required. In an effort to achieve low- to mid- throughput purification (1-16 plates in an eight-hour workday), we have performed initial experiments to integrate the Eppendorf Perfectprep BAC 96 kit onto the Caliper Life Sciences Sciclone ALH 3000 to provide an automated 96 well BAC purification system.
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.
- 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 tips, 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.
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:
The 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.
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.