1. Dispense AMPure XP beads into PCR tubes

Transfer AMPure XP beads from the stock solution into 12 PCR tubes placed in a cooling block from INTEGRA.
Note: The cooling block is just used as a support in this instance, not for cooling down the samples.

To ensure a homogenous stock solution, beads are thoroughly mixed by shaking/inverting until the solution appears consistent in color. The beads are transferred into 12 PCR tubes using the Repeat Dispense mode of a VIAFLO single channel 1250 μl electronic pipette. A customized VIALINK program (AMP_Transfer1) is available to aid bead transfer.

For optimal pipetting, ensure beads are thoroughly mixed before each transfer. Mixing steps can be defined by the number of cycles and the pipetting speed. Both influence the efficiency of mixing and thus the quality of the clean-up. Saving these parameters in the pipetting program ensures that mixing is always carried out as defined, yielding consistent results. Insert a pre- and post-dispense step to enhance accuracy and precision while pipetting precious reagents, such as AMPure XP beads.

Tip: The use of Sterile, Filter, Low Retention GRIPTIPS ensures that every dispense is as accurate as possible, with no loss of beads or sample.

2. Transfer AMPure XP beads into the DNA samples

Transfer AMPure XP beads from the PCR tubes into a 96 well plate preloaded with DNA samples.

Pipette the beads from the PCR tubes into the 96 well plate using a VIAFLO 12 channel 50 μl electronic pipette. For optimal pipetting, make sure the tips are exchanged, and mix the beads thoroughly before each transfer. A customized VIALINK program (AMP_Transfer2) is provided for this step.

Tip: Use Low Retention GRIPTIPS to minimize loss of beads adhering to the tip wall.

3. Mixing and binding of the AMPure XP beads

Mixing and binding of the magnetic beads to the PCR samples.

Load GRIPTIPS (position A) then select and run the AMPure_XP_M program on the VIAFLO 96. The samples are now mixed 10 times by pipetting up and down on position B. A five minute wait time follows, timed by the VIAFLO 96, to allow the DNA to bind to the beads.

Tip: Use the z-height setting of the VIAFLO 96 to define the optimal tip immersion depth. This prevents air entering the tip during mixing and avoids the pipette tip touching the bottom of the plate. Setting the Tip Align support strength to 3 for positions A and B makes it more comfortable to use the VIAFLO 96. These settings can be incorporated into the program so that they are not forgotten.

4. Magnetic separation of the AMPure XP beads

Separating the magnetic beads from the PCR samples.
Note: Make sure new GRIPTIPS are loaded before continuing the protocol to ensure removal of the supernatant without bead carryover.

A prompt on the pipette screen reminds the user to move the sample plate from position AB onto the 96 well magnet (position B) and place an automation friendly reagent reservoir for waste collection on position AB. After a two minute incubation time the beads form a ring-shaped structure and the solution becomes clear. Load new GRIPTIPS before continuing the procedure to ensure accurate removal of the supernatant without bead carryover. Follow the instructions on the pipette and aspirate the supernatant slowly from the sample, dispensing it into the waste reagent reservoir (position AB).

Tip: To avoid disturbing the ring of beads the supernatant is aspirated slowly at speed 1. Leave 5 μl of supernatant in the plate to prevent beads being drawn out during aspiration. The z-height limit is again used to ensure that the beads are not disturbed during pipetting.

5. AMPure XP bead clean-up

Wash the magnetic beads twice with 70 % ethanol.

Place an automation friendly reagent reservoir containing 70 % ethanol on position A and change the GRIPTIPS before continuing with the wash step. Follow the prompts on the pipette. Pre-wet the GRIPTIPS with 70 % ethanol. Then wash the samples with 70 % ethanol. Repeat the washing step again as indicated by the pipette.

Tip: Pre-wetting the GRIPTIPS with 70 % ethanol ensures equilibration of the humidity and the temperature between the air in the pipette/tips and the sample/liquid. In-house testing has shown that Low Retention GRIPTIPS prevent ethanol from dripping while traveling from one pipetting position to another.

6. Elute samples from the magnetic beads

Elute the purified samples from the magnetic beads by adding the elution buffer.

As indicated by the pipette, replace the 70 % ethanol reagent reservoir on position A with an elution buffer reagent reservoir and move the sample plate from the magnet (position B) to position AB. Load new GRIPTIPS before continuing with the protocol. After transferring and thoroughly mixing the elution buffer with the beads, the pipette prompts the user to place the sample plate back onto the magnet (position B). During the one minute incubation time, place a new 96 well plate on position AB.

7. Transfer the sample eluates

Transfer the sample eluates into the new 96 well plate.
Note: Load new GRIPTIPS to ensure a clean eluate transfer without bead carryover.

Continue with the same program, slowly and carefully transferring the eluates from position B into the new plate (position AB).

Tip: Optimizing pipette settings (aspiration speed, volume and height) allows the volume of the transferred eluate to be maximized without carryover of beads. These settings can be easily tweaked at any time. Performing a test run with water before implementing any new assay is an ideal way to optimize pipette settings.

Remarks

Automatic mode:

The VIAFLO 96 can also operate on its own, enabling less user interaction which in turn improves ergonomics and reproducibility. This is also making is even more ideal for use in tight spaces, such as under a laminar flow cabinet.

Partial tip load:

If you are not working with a full set of 96 samples, the VIAFLO 96 is able to work with any number of tips loaded, allowing purification of smaller numbers of samples.