Overview: How to automate MACHEREY-NAGEL's NucleoSpin Plasmid Transfection-grade kit

The ASSIST PLUS pipetting robot is used to automate the pipetting steps of the MACHEREY-NAGEL NucleoSpin 96 Plasmid Transfection-grade kit purification protocol. The liquid handling platform guides the user whenever manual interventions are required during the process. The following procedure is based on the kit manufacturer’s protocol for purification of 96 samples.

Bacteria are first cultivated at 37 °C following MACHEREY-NAGEL’s recommendations, either in a square-well block or tubes. For the present protocol, the bacterial cultures are grown in tubes and transferred into a square-well block. The cultures are centrifuged for 10 min at 1000 x g to pellet the bacteria. The supernatant is discarded, and the residual medium removed by tapping the plate upside down on a clean paper sheet or soft tissue.

The ASSIST PLUS pipetting robot operates a VIAFLO 12 channel 1250 µl electronic pipette with 1250 µl Sterile, Filter GRIPTIPS®.

Downloads: App note and protocols for the Transfection-grade plasmid DNA purification

Here you can download the complete protocols and reports used in this application and use them on your ASSIST PLUS. The protocol can be customized with the VIALAB software.

Experimental set-up

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

Deck position A: 8 row reagent reservoir containing the different buffers for the protocol (Figure 3).

Deck position B: 96 well culture plate (square-well block) containing the centrifuged bacterial cells placed on the Teleshake microplate shaker in portrait orientation.

Deck position C: NucleoVac 96 Vacuum Manifold containing and/or supporting the different 96 well plates. The vacuum manifold needs to be placed on the instrument in portrait orientation.

IMPORTANT: Make sure that the vacuum manifold outlet is positioned towards the user, so that the tower of the pipetting robot can move freely along the x-axis. Also check that the Teleshake cable does not interfere with the tower movement.

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) in it.

1. Resuspension of the bacterial cells

Resuspend the harvested bacterial cells.

Fill the 8 row reagent reservoir with the different buffers as shown in Figure 3. Select and run the VIALAB program ‘MN Plasmid TG’. The ASSIST PLUS transfers 250 µl of 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 600 rpm. The VIALAB program includes a 4 minute delay, after which the pipette informs the user to stop shaking the plate.

2. Lysis of the bacterial cells

Lyse the suspended bacterial cells.

The ASSIST PLUS transfers 250 µl of Buffer A2 from row B of the reservoir into the cell culture plate using the Repeat Dispense function. Turn on the shaker as indicated by the pipette, and incubate at room temperature with moderate shaking (300 rpm). A 2 minute delay is set in the VIALAB program, after which the pipette informs the user to stop shaking the plate. The program then continues directly with the next step.

Tip:

• Introducing a tip touch on the upper right side of the wells allows the pipette tips to be reused while avoiding contamination of the source.

3. Neutralization

Neutralize the lysis buffer.

The ASSIST PLUS pipetting robot adds 350 µl of Neutralization Buffer A3 to the suspension using the Repeat Dispense mode.

Tip:

• For lysis in the plate, mix by pipetting up and down either after adding the buffer or before loading onto the NucleoSpin Plasmid Filter Plate. The VIALAB program can be easily adapted to introduce a mix cycle at the end of each dispense of the Neutralization Buffer A3.

4. Lysate clearing

Clear the crude lysates by vacuum filtration using the NucleoSpin Plasmid Filter Plate.

The suspension is mixed twice by pipetting the whole volume up and down. The entire volume is then transferred to the NucleoSpin Plasmid Filter Plate. A 1 minute delay is set to allow room temperature incubation for optimal precipitation. The pipette prompts the user to turn on the vacuum pump. Apply a vacuum of -0.2 to -0.4 bar and adjust it to establish a flow rate of 1-2 drops per second (this takes 4 minutes, including a delay set up in the VIALAB program). When the crude lysate has passed through the NucleoSpin Plasmid Filter Plate, release the vacuum as indicated by the pipette.

Tip:

• The use of Wide Bore GRIPTIPS may prevent shearing of DNA when transferring the crude lysate to the NucleoSpin Plasmid Filter Plate.

5. DNA binding

Bind DNA to the silica membrane.

Remove and discard the NucleoSpin Plasmid Filter Plate. Open the manifold lid and remove the NucleoSpin Plasmid Binding Plate containing the cleared lysates. Insert the Wash Plate onto the spacers inside the manifold base, replace the lid on the base and put the NucleoSpin Plasmid Binding Plate on top of the manifold. The vacuum manifold is now 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 completely drained, then release the vacuum.

6. Washing and drying

Wash and dry the silica membrane.

The ASSIST PLUS adds 900 µl Buffer ERB (detoxification buffer) 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 Buffer AQ (Figure 5). Release the vacuum. The ASSIST PLUS transfers 900 µl of Buffer AQ 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 Buffer AQ 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 clean paper sheet supplied. Remove the MN 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) for 12 min to dry the membrane completely, and to remove any trace of ethanol that may inhibit subsequent enzymatic reactions. Maintaining a continuous air flow is the most important aspect for this step.

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

Elute the bacterial DNA.

Place the U-bottom elution plate 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 (-0.4 to -0.6 bar) for 1 minute, release it, then remove the elution plate containing the DNA and seal it for storage.

Tip:

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

Results

The validation data show high quality plasmid DNA from three different constructs with different plasmid sizes on one plate using the ASSIST PLUS pipetting robot.

Remarks

VIALAB software:

The VIALAB programs can be easily adapted to your specific labware and protocols, for instance, if lysis of the bacterial cells is done in tubes.

Partial plates:

Programs can be adapted to a different number of samples at any time, giving laboratories total flexibility to meet current and future demands.