Overview: How to get ultra pure DNA with Zymo's DCC kit

Step-by-step procedure

The ASSIST PLUS pipetting robot is used together with the 8 channel 300 µl VOYAGER automated tip spacing electronic pipette and 300 µl sterile, filter, low retention GRIPTIPS in this semi-automated workflow to purify nucleic acid (Figure 1).

Downloads: App note and protocols for automated DNA clean-up using Zymo Research DNA Clean & Concentrator MagBead Kit

The total DNA clean-up workflow is composed of 3 main steps:

1. Binding
2. Washing
3. Elution

Experimental set-up

Deck position A: dual reservoir adapter with 2 divided reservoirs

Deck position B: Sapphire 96 well PCR plate (Greiner Bio-one) on a PCR cooling block

Deck position C: Thermo Scientific™ Abgene™ 96 well 0.8 ml polypropylene DeepWell™ plate

Binding

Preparation of samples, buffer and magnetic bead addition.

Place the dual reservoir adapter at deck position A. Place 2 divided reservoirs onto the deck, fill 1 of the 10 ml compartments with 2.2 ml of DNA MagBinding Buffer (Figure 2, pink) and the other with 9 ml of DNA Wash Buffer (Figure 2, lilac). Next, place a 96 well PCR plate with nuclease-free water, MagBinding Beads and DNA Elution Buffer (Figure 2, blue, green and yellow, respectively) at position B (Figure 3).

Lastly, place the DeepWell plate containing the 30 µl samples (Figure 2, dark blue) at deck position C.

Select and run the VIALAB program 'Zymo DCC'. The VOYAGER automated tip spacing electronic pipette will transfer 20 µl of nuclease-free water (with a 5 µl pre- and post-dispense) into the samples, so that the starting volume of the nucleic acid to be purified is 50 µl, as suggested by the DCC kit manufacturer. Next, the correct volume of DNA MagBinding Buffer – corresponding to 4 times the volume of input DNA so, in this case, 4x50 μl=200 μl – is added to each sample, followed by 10 cycles of accurate mixing at speed 5. The MagBinding Beads are mixed thoroughly over 10 cycles to ensure they are kept in suspension, and 20 µl of beads are added to the samples (Figure 4). The magnetic beads are then mixed with the nucleic acid to be purified over 15 cycles.

In the next step, the pipetting robot instructs the user to put the DeepWell plate onto the shaker for 10 minutes at 1300 rpm. This step improves the binding of the nucleic acid to the magnetic beads.

After shaking, a message on the pipette will tell the user to put an empty reservoir at deck position A (A2) for waste and to place the DeepWell plate with the sample and magnetic beads onto the magnetic separation device at deck position C (Figure 5).

After pressing ‘OK’, a 2-minute incubation will follow to allow magnetic bead capture. The pipetting robot will then automatically move on to the next step, where it will remove the supernatant from the plate on the magnetic separation device.

Tips:

• Pre- and post-dispense steps can be used in liquid transfers throughout this protocol to guarantee precise pipetting.
• Use slow aspiration speeds of 1 or 2 during supernatant removal to avoid magnetic bead loss.

Washing

Purification with DNA Wash Buffer.

The pipette directs the user to remove the magnetic separation device and place the DeepWell plate back at position C. In the first purification step, 500 µl of DNA Wash Buffer is added in 2 transfer steps and mixed with the magnetic beads (Figure 6). Then a message on the pipette tells the operator to place the DeepWell plate back onto the magnetic separation device at position C and incubate it for 2 minutes. After incubation, the pipetting robot goes directly to the next step and removes the supernatant. The first washing step is repeated, and then the leftover wash buffer is carefully removed. The pipette informs the user that a 10 - minute incubation time is now required to dry the magnetic beads; this incubation step is needed to ensure that residual buffer does not inhibit downstream applications.

Tip:

• The wash buffer contains ethanol. Make sure you use the correct pipetting speed, air gap, and low retention tips for precise pipetting without dripping.

Elution

Eluting the samples in DNA Elution Buffer.

After the drying step, the operator should remove the magnetic separation device from below the DeepWell plate. ASSIST PLUS will add 50 µl of DNA Elution Buffer and then perform 10 mixing cycles (Figure 7). A message will pop up, telling the user that the DeepWell plate should be placed onto the shaker for 5 minutes. This guarantees that all the DNA is eluted from the magnetic beads into the buffer. After shaking, the DeepWell plate should be placed onto the magnetic separation device at position C, where the magnetic beads will be captured during a 2-minute incubation. The pipetting robot will then inform the user that a new 96 well plate for the eluted DNA should be placed at deck position B in landscape orientation. In the last step, the pipette transfers 45 µl of eluted DNA to the new 96 well plate.

Tip:

• The eluted DNA can be used immediately or stored at -20 °C until further use.

Results

The automated DCC MagBead protocol on ASSIST PLUS shows high DNA recovery from PCR,  that is ready to use in any downstream application. The automated workflow resulted in greater DNA yield, better purity (A260/A280 and A260/A230 ratios) and higher reproducibility (lower SD values).

Remarks

• VIALAB software: VIALAB programs can be easily adapted to the user’s specific labware and protocols.
• Run report: If the ASSIST PLUS pipetting robot is connected to the PC with VIALAB, programs can be started directly from the PC. A report is automatically generated after a run, documenting details such as the start/end time, user, calculated volumes and any errors that occurred. This offers a convenient way to fulfill regulatory requirements.