Plasmid DNA purification of MACHEREY-NAGEL’s NucleoSpin® 96 Plasmid kit and NucleoVac 96 Vacuum Manifold

  • Plasmid DNA purification of MACHEREY-NAGEL’s NucleoSpin® 96 Plasmid kit and NucleoVac 96 Vacuum Manifold

    Semi-automated miniprep protocol on the ASSIST PLUS pipetting robot

    The use of silica membrane-based DNA purification kits is a convenient way to prepare plasmid DNA samples for cloning or subsequent analysis, e.g. sequencing or restriction analysis. MACHEREY-NAGEL’s NucleoSpin 96 Plasmid kit is designed for high throughput purification of high-copy plasmid DNA from E. coli in a 96 well plate format. The alkaline lysis-based miniprep protocol typically yields 5-15 μg of plasmid DNA from 1.5ml overnight cultures. However, it is a time-consuming step in genetic analysis. Automation of the protocol on an ASSIST PLUS pipetting robot with a VIAFLO 12 channel 1250 μl electronic pipette offers an easy and efficient way to extract and purify plasmid DNA with minimal hands-on time, guaranteeing perfect and reproducible liquid handling while protecting the user from repetitive strain injuries.

  • Table of contents

    The use of silica membrane-based DNA purification kits is a convenient way to prepare plasmid DNA samples for cloning or subsequent analysis, e.g. sequencing or restriction analysis. MACHEREY-NAGEL’s NucleoSpin 96 Plasmid kit is designed for high throughput purification of high-copy plasmid DNA from E. coli in a 96 well plate format. The alkaline lysis-based miniprep protocol typically yields 5-15 μg of plasmid DNA from 1.5ml overnight cultures. However, it is a time-consuming step in genetic analysis. Automation of the protocol on an ASSIST PLUS pipetting robot with a VIAFLO 12 channel 1250 μl electronic pipette offers an easy and efficient way to extract and purify plasmid DNA with minimal hands-on time, guaranteeing perfect and reproducible liquid handling while protecting the user from repetitive strain injuries.

Key benefits

  • MACHEREY-NAGEL’s NucleoSpin 96 Plasmid kit and NucleoVac 96 Vacuum Manifold are a proven method for high throughput plasmid DNA purification.
  • Automation of the pipetting steps of the miniprep workflow with the ASSIST PLUS pipetting robot allows for more hands-free time for the user and increased reproducibility.
  • 96 samples can be purified in less than 1 hour by processing 12 samples in parallel. In combination with the ASSIST PLUS, the VIAFLO electronic pipettes provide unmatched pipetting ergonomics, eliminating the need to hold the pipette during the pipetting steps.
  • The ASSIST PLUS performs all the pipetting steps of the protocol and guides the user through each manual intervention.

Overview

The ASSIST PLUS pipetting robot is used to automate the pipetting steps of the MACHEREY-NAGEL NucleoSpin 96 Plasmid kit protocol for plasmid DNA purification. The liquid handling platform guides the user whenever manual interventions are required during the process. The following procedure is an example 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 MACHEREY-NAGEL Culture Plate (Square-well Block) or in tubes. For the current protocol, the bacterial cultures are grown in tubes and transferred into a Culture Plate. Centrifuge the bacterial cultures for 10 min at 1000 x g to pellet the bacteria. Discard the supernatant and remove the residual medium 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: Protocols and reports for automated plasmid DNA purification with ASSIST PLUS

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.

Deck position B:

96 well Culture Plate 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: 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.

Figure 1: General set-up for the plasmid DNA purification program. Position A: 8 row reagent reservoir. Position B: 96 Square-well Block placed on the Teleshake microplate shaker. Position C: NucleoVac 96 Vacuum Manifold.
Figure 1: General set-up for the plasmid DNA purification program. Position A: 8 row reagent reservoir. Position B: Culture Plate (96 Square-well Block) placed on the Teleshake microplate shaker. Position C: NucleoVac 96 Vacuum Manifold.

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.

Figure 2: Vacuum manifold set-up for steps 1 to 4. Insert the MTP/Multi-96 Plate spacer, waste container and NucleoSpin Binding Plate (white rings, left) in the manifold. Place the manifold lid on top of the manifold base, and then place the NucleoSpin Plasmid Filter Plate (violet rings, right) on top of the manifold.
Figure 2: Vacuum manifold set-up for steps 1 to 4. Insert the MTP/Multi-96 Plate spacer, waste container and NucleoSpin Binding Plate (white rings, left) in the manifold.

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.
Figure 3: Set-up for the plasmid DNA purification program. Position A: 8 row reagent reservoir filled with Resuspension Buffer A1 in row A (blue, 26 ml), Lysis Buffer A2 in row B (purple, 25 ml), Neutralization Buffer A3 in rows C and D (yellow, 18 ml) and Wash Buffer AW in rows E and F (green, 30 ml). Position B: 96 Square-well Block containing the centrifuged bacterial cells for resuspension (in pink), placed on the Teleshake. Position C: NucleoVac 96 Vacuum Manifold set up as described in Figure 2.
Figure 3: Set-up for the plasmid DNA purification program.

2. Lysis of the bacterial cells

Lyse the suspended bacterial cells.

The ASSIST PLUS transfers 250 μl of Lysis 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 directly continues with the next step.

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.

4. Clearing of the crude lysates

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

The suspension is mixed three times by pipetting up and down, and then the entire volume is 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 approx. 4 minutes, including a delay set up in the VIALAB program). When the crude lysate has passed 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 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.

Figure 4: Vacuum manifold set-up for steps 5 and 6. The MN Wash Plate is inserted on the spacers inside the manifold base (left). The NucleoSpin Plasmid Binding Plate (white rings) with the cleared lysates is placed on top of the manifold (right).
Figure 4: Vacuum manifold set-up for steps 5 and 6. The Wash Plate is inserted on the spacers inside the manifold base (left). The NucleoSpin Plasmid Binding Plate (white rings) with the cleared lysates is placed on top of the manifold (right).

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.
Figure 5: Set-up for the second and third wash steps. The 8 row reagent reservoir in Position A is filled with Wash Buffer A4 in rows A to H (purple, 23 ml). In Position C, the wells in pink indicate the presence of bacterial DNA bound onto the silica membrane of the NucleoSpin Binding Plate.
Figure 5: Set-up for the second and third wash steps. The 8 row reagent reservoir in Position A is filled with Wash Buffer A4 in rows A to H (purple, 23 ml).

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.
    Figure 6: Vacuum manifold set-up for the elution step. The Elution Plate U-bottom is inserted on the spacers inside the manifold base (on the left side). The NucleoSpin Plasmid Binding Plate (white rings) with the cleared lysates is placed on top of the manifold (on the right side).
    Figure 6: Vacuum manifold set-up for the elution step. The Elution Plate is inserted on the spacers inside the manifold base (left). The NucleoSpin Plasmid Binding Plate (white rings) with the cleared lysates is placed on top of the manifold (right).
    Figure 7: Set-up for the elution step. The 8 row reagent reservoir in Position A is filled with Elution Buffer AE in row H (blue, 13 ml). Position C: Bacterial DNA bound on the silica membrane of the NucleoSpin Binding Plate (pink).
    Figure 7: Set-up for the elution step. The 8 row reagent reservoir in Position A is filled with Elution Buffer AE in row H (blue, 13 ml). Position C: Bacterial DNA bound on the silica membrane of the NucleoSpin Binding Plate (pink).

    Remarks

    VIALAB software:

    The VIALAB programs can be easily adapted to your specific labware and protocols, for instance when partial plates are needed, or if lysis of the bacterial cells is performed in tubes.

    Partial plates:

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

    Conclusion

    • The MACHEREY-NAGEL NucleoSpin 96 Plasmid kit protocol for plasmid DNA purification can be easily automated with the ASSIST PLUS pipetting robot and a VIAFLO 12 channel 1250 μl electronic pipette. Using the NucleoVac 96 Vacuum Manifold directly on the deck allows for a compact set-up for processing up to 96 samples in one run.
    • All the pipetting steps are automated to guarantee maximum reproducibility and consistency of the results, as well as optimal ergonomics for the user to avoid repetitive strain injuries.
    • Together with the VIAFLO electronic pipette, the ASSIST PLUS pipetting robot acts as a trusted laboratory assistant and guides the user through the whole protocol, ensuring an error-free workflow.
    • The entire purification protocol is included in one single VIALAB program that can be rapidly modified to meet your specific needs.

    September 2020, by

    • Jan-Peter Baldin, PhD

      Product Manager

      Email

    Any questions? I'm happy to help!

    • Stephanie Stursberg

      Product Manager

      Email

    Used instruments and accessories

    ASSIST PLUS, Pipetting Robot VIAFLO 12 channel, 1250 µl, electronic pipette GripTip, 1250 µl, Sterile, Filter (for automation systems) 8 Row Polypropylene Reservoirs (6372) INHECO: Teleshake MACHEREY‑NAGEL: Culture Plate MACHEREY‑NAGEL: NucleoSpin 96 Plasmid, 96‑well kit for plasmid DNA MACHEREY‑NAGEL: NucleoVac 96 Vacuum Manifold VACUUBRAND: Vacuum Pump

    ASSIST PLUS, Pipetting Robot

    INTEGRA has developed the ASSIST PLUS pipetting robot to streamline routine pipetting tasks at an affordable price. Using INTEGRA electronic multichannel pipettes, the system:

    • automates pipetting tasks,
    • eliminates physical strain and
    • ensures superior reproducibility and
    • error free pipetting.

    Part No. 4505

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    ASSIST PLUS Pipetting Robot

    VIAFLO 12 channel, 1250 µl, electronic pipette

    The Touch Wheel is a quick and ergonomic way to modify pipetting parameters. Instead of repeatedly pushing buttons or twisting fingers to modify volumes, you simply slide your finger over the wheel.

    The full color screen provides full text menus (in multiple languages) and displays pipetting protocols without abbreviations, making VIAFLO pipettes particularly easy to understand and intuitive to use.

    Part No. 4634

    Find out more about VIAFLO electronic pipettes
    INTEGRA’s range of VIAFLO electronic pipettes.

    GripTip, 1250 µl, Sterile, Filter (for automation systems)

    GripTip pipette tips perfectly match the multi-lobe tip fitting, snapping firmly on during loading. This guarantees a perfect seal on every tip, preventing them from loosening, leaking or completely falling off. All tips are precisely aligned horizontally, enabling accurate touch-offs, even when pipetting with 384 tips.

    Part No. 6445

    Learn more about GripTips pipette tips for Benchtop Pipetting Systems
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    8 Row Polypropylene Reservoirs (6372)

    The polypropylene design improves the reservoirs chemical compatibility and also allows the reservoirs to be autoclaved.

    • Reagent reservoirs for 96 well plates
    • Pyramid bottom
    • Each row holds 32 ml

    Part No. 6372

    Show all automation-friendly reagent reservoirs
    8 Row Reagent Reservoirs Blocks, Non-Sterile, Sterile

    INHECO: Teleshake

    The Teleshake and Teleshake 1536 have a very compact and low-profile design with a height of only 39 and 56mm, respectively. The small footprint makes them ideal for integration into automation platforms. Plates with up to 384 wells can be used on the Teleshake while the Teleshake 1536 is ideal for plates with 384 up to 1536 wells that need higher shaking frequency.

    Source: Website INHECO

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    INHECO Teleshake

    MACHEREY‑NAGEL: Culture Plate

    96‑well blocks with 2.1 mL square wells, including Gas-permeable Foils.

    Source: Website MACHEREY-NAGEL

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    Culture Plate

    MACHEREY‑NAGEL: NucleoSpin 96 Plasmid, 96‑well kit for plasmid DNA

    Rapid Mini preparation of plasmid DNA in proven 96‑well format.

    Source: Website MACHEREY-NAGEL

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    NucleoSpin 96 Plasmid, 96‑well kit for plasmid DNA

    MACHEREY‑NAGEL: NucleoVac 96 Vacuum Manifold

    Vacuum manifold consists of manifold base and lid, a spacer set, and two waste containers.

    Source: Website MACHEREY-NAGEL

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    NucleoVac 96 Vacuum Manifold

    VACUUBRAND: Vacuum Pump

    Chemistry-design diaphragm pumps are an excellent solution for continuous, oil-free pumping of corrosive gases and vapors.

    Source: Website VACUUBRAND

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    Customer’s voice

    SAVED OUR LIVES! Absolutely love our Assist Plus (We lovingly call her EVA from the movie Wall-e)! She has made our lives so much easier and saved my pipetting hand from all the high-throughput assays we run!

    Mariam Iftikhar, Omniome