High-throughput isolation of ultra-pure plasmid DNA by a robotic system

Background With the availability of complete genomes, a systematic inventory of cellular processes becomes achievable. This requires assessing the function of all individual genes. Transfection of plasmid DNA into cell culture cells is an essential technique for this aim as it allows functional overexpression or downregulation of genes. While many robotic systems isolate plasmids for sequencing purposes, for more demanding applications such as transfections there is a shortage of robots for the high-throughput isolation of plasmid DNA. Results Here we describe a custom-made, automated device, which uses a special protocol to isolate plasmid DNAs with a purity sufficient for efficient transfections into mammalian cells. Approximately 1,600 ultra pure plasmids can be isolated in a 96-well plate format within 12 hours. As a unique feature the robot comprises the integration of a centrifuge instead of expensive columns, the use of a custom-made pipetting head with a movable gripper, especially designed shaking platforms and an acetone wash facility. Conclusion Using this robot we demonstrate how centrifugation steps with multiple precipitations, most notably through a precipitation step of SDS in isopropanol, lead to high purity plasmid DNA and make possible high-throughput transfections into mammalian cells for functional gene annotations.

is much faster compared with the first operation mode but less accurate. It is used in the process for washing the pipetting tips.

Gripper
The gripper is mounted directly behind the movable pipettor. The gripping hand ((6), Fig. S2) is driven by an HGP-16-A pneumatic element (Festo AG, Esslingen, Germany). A microswitch sensor detects whether a DWP has been taken up. The gripping hand is mounted on the bottom end of a pneumatic linear unit (SLG-12-300-P-A, Festo AG, (7)) which can move the gripping hand 20cm down from its top position. The exact z base position for taking up a DWP is adjusted using the Z1 sled, with the pneumatic linear unit in top position. From there the linear unit moves the hand 20cm down to the actual pick-up point on the platform.

Shaking platforms
Six shaking platforms are available on the work area of the robot. They are used both as static platforms and as linear shakers for the DWPs (Fig. S3 and S4). The static part mounted on the base plate (2) is shown in red colour. During loading, unloading and static use the shaking cylinder (1), which is activated by the valve (4), holds the shaking platform (11) in the left position that is exactly defined by the delimiter (7,7a). The rotating locks (17) are released (open) and the microswitch (5, 5a) signals "loading coordinates ok". Positioning of the DWPs is facilitated by the guiding frames (12). For shaking, the piston of the cylinder (1), which is connected to the shaking part by (8), moves the platform (11) periodically forward and backward between the stops of the delimiter (7,7a). The moving part is guided by rolls (10), which are mounted on the frame (9) and move on the shaking rail (6). With the  Figure   S5 shows the hardware controllers for a pair of

Fig. S12: The VEE program function (subroutine), which starts the centrifuge under imbalance control with error handling (A-D). Restart of the centrifuge after imbalance detection and manual DWP re-adjustment by the system supervisor (E, F). Inputs to the function (ASCII strings) at the left hand side strip: (A) centrifugation time; (B) revolutions/min
Pipetting station S1/S2 All three pipetting heads (S1a, S1b, S2a) of the independent pipetting station S1/S2 (Fig.   S13) work according to the "suction-pressure" principle 1 (Fig. S14) in which suction and pressure are generated in a common air tube and relayed to the 96 pipetting tips. This requires that the pipetting tips are selected for uniform outlet openings. Fig. S15 explains how tips are selected from mixed sizes delivered by the manufacturers. Air flows from the pump (P) through the throttle (th) and the tip to be tested which is plugged on the test cone (TC).
Depending on the flow resistance of the tip, which is determined by the size of its opening, the digital pressure meter (DM) measures a characteristic pressure value. The device is controlled by measuring a reference tip ("Ref") in certain time intervals. If its pressure value has changed the initial reference pressure value has to be restored by adjustment of the throttle. The tested tips are classified into pressure groups, which constitute groups of equal openings. Lower pressures correspond to larger openings and vice versa. The mean opening size of the tips has to be considered for the determination of the pipetting volume when Fig. S13: The separate pipetting station S1/S2. The pipetting heads (S1a), (S1b/S2a) can swing out. The platforms S1 and S2 are then availabe for loading. The gripper (Gp) mounted on the arm (Gpa) loads a DWP to platform S2. The lid (Al) of the cooled acetone vessel (Ac) is closed. The collectors S1w and S2w take up the waste from the pipettors S1b and S2a. The main pipettor (Pk) is moved with the gripper without a functional role in the S1/S2 pipetting station. The 96-tip pipetting heads S1a and the joined heads S1b and S2a (Fig.   S13) swing out when a DWP is delivered by the gripper (Gp,Gpa). the DWP is also placed on platform S1, pipettor S1b is swung over the DWP, moved down until the tips are immersed into the acetone supernatant and the supernatant is taken up by a short suction pulse. S1b is moved up, swung out over the waste S1w and the content of the pipetting tips is blown out.
The number of suctions, the depth of immersion and the strength and duration of the suction pulses are determined experimentally. The complete acetone washing process (Fig. S16) is performed in one cycle that is run twice: Addition of 400µl acetone to the DWPs, transport to  A3/B3  platform,  shaking,   transport  to  centrifuge, centrifugation, transport to S1, removal of acetone supernatant and addition of 400µl acetone.

Wash station for pipetting head
Each pipetting step of the main pipetting head is followed by washing in the tip washing station (Fig. S17)  Field III offers a collection of functions for manual execution, e.g. testing when the main processes are not executed.