Experimental set-up

Prepare the deck of the pipetting robot as follows (Figure 1):

Important: Align the Teleshake and vacuum manifold before each run (see appendix in the PDF). The plasmid DNA purification protocol is performed using a pipetting program generated with the VIALAB software. The ASSIST PLUS liquid handling platform guides the user through each step of the protocol.

Overview of the extraction and purification steps:

1. Resuspension of the bacterial cells
2. Lysis of the bacterial cells
3. Neutralization
4. Lysate clearing
5. DNA binding
6. Washing and drying
7. Elution

For the initial set-up, assemble the manifold as described in Figure 2, with the NucleoSpin Plasmid Filter Plate (violet rings) on top of the manifold and the NucleoSpin Binding Plate (white rings) inside.

1. Resuspension of the bacterial cells

Resuspend the harvested bacterial cells.

Fill the 8 row reagent reservoir with the different buffers as described in Figure 3. Select and run the VIALAB program ‘MN Plasmid’. The ASSIST PLUS transfers 250 μl of Resuspension Buffer A1 from row A of the reservoir into the Culture Plate using the Repeat Dispense mode. Turn on the shaker as indicated by the pipette and resuspend the cells by shaking at 1000 rpm. The VIALAB program includes a 4 minute delay, after which the pipette informs the user to stop shaking the plate.

Tips:

• Adding an air gap at the end of the aspiration step eliminates droplet build-up after the liquid dispense and avoids the use of a tip touch to prevent any risk of cross-contamination.
• For the same reason, the ASSIST PLUS also dispenses the buffer to the upper part of the wells without tip travel.
• Adding a post-dispense ensures the accuracy of each dispense when using the Repeat Dispense mode.

5. DNA binding

Bind DNA to silica membrane.

Remove and discard the NucleoSpin Plasmid Filter Plate. Open the manifold lid and remove the NucleoSpin Plasmid Binding Plate (white rings) containing the cleared lysates. Insert the Wash Plate on the spacers inside the manifold base, replace the lid on the base and place the NucleoSpin Plasmid Binding Plate on top of the manifold. The vacuum manifold is ready for the next step (Figure 4). Apply the vacuum (-0.2 to -0.4 bar, 1 min, flow rate of 1-2 drops per second) until the cleared lysate has drained off, then release the vacuum.

6. Washing and drying

Wash and dry the silica membrane.

The ASSIST PLUS adds 600 μl Wash Buffer AW to each well. After a 30 second incubation, it informs the user to apply a vacuum (-0.2 to -0.4 bar, 1 min, flow rate of 1-2 drops per second). In the meantime, prepare an 8 row reagent reservoir filled with Wash Buffer A4 (Figure 5). Release the vacuum. The ASSIST PLUS transfers 900 μl of Wash Buffer A4 containing ethanol to each well for a second wash step. Using the same conditions as before, apply the vacuum after incubation, release it, and allow the pipette to transfer 900 μl of Wash Buffer A4 to each well for the third wash step. Apply the vacuum after incubation (same settings as before). Remove any residual wash buffer from the NucleoSpin Plasmid Binding Plate and tap the outlets of the plate onto the supplied clean paper sheet. Remove the Wash Plate and the waste container from the manifold base and place the NucleoSpin Binding Plate on top of the manifold. Apply a vacuum ( 0.4 to -0.6 bar; maintaining a continuous air flow is the most important aspect for this step) for at least 10-15 min to dry the membrane completely and remove any trace of ethanol that may inhibit subsequent enzymatic reactions.

Tips:

• Pre-wetting the tips prior to pipetting and having an air gap at the end of the aspiration prevents droplets and dripping when pipetting volatile liquids such as ethanol. Additionally, Low Retention GRIPTIPS can be used for these pipetting steps.
• The pipetting speeds have been set up specifically according to the nature of the buffers.

7. Elution

Eluate the bacterial DNA.

Place the Elution Plate (U-bottom) in the manifold base and the NucleoSpin Plasmid Binding Plate on top of the manifold (Figure 6). Place an 8 row reservoir containing Elution Buffer AE in row A on Position A (Figure 7). The ASSIST PLUS pipetting robot dispenses 150 μl Elution Buffer AE into the Binding Plate. After a 2 minute incubation period, apply a vacuum for 1 minute ( 0.4 to -0.6 bar), release the vacuum, then remove the Elution Plate containing the eluted DNA and seal it for further storage.

Tip:

• When another DNA concentration is required, simply adapt the elution volume according to MACHEREY NAGEL’s recommendations using the VIALAB Software.