PCR purification with Beckman Coulter AMPure XP magnetic beads and the VIAFLO 96

  • PCR purification with Beckman Coulter AMPure XP magnetic beads and the VIAFLO 96

    Automatic magnetic bead purification with the VIAFLO 96 handheld electronic pipette

    Agencourt AMPure XP magnetic beads (Beckman Coulter) are an efficient way to clean up samples for PCR, NGS, cloning and microarrays. The kit provides a solution for medium to high throughput requirements when carried out in a 96 well plate, but the protocol involves many washing and transfer steps that make it tedious to perform manually. With the VIAFLO 96, a handheld 96 channel electronic pipette, multistep protocols such as PCR clean-up and DNA purification can be performed quickly and efficiently, increasing throughput tremendously by transferring samples and reagents to all 96 wells at once. Thanks to its unique operating concept, the VIAFLO 96 remains as easy to use as a traditional handheld pipette and can even provide critical information (user-defined prompts) about the protocol steps.

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      Agencourt AMPure XP magnetic beads (Beckman Coulter) are an efficient way to clean up samples for PCR, NGS, cloning and microarrays. The kit provides a solution for medium to high throughput requirements when carried out in a 96 well plate, but the protocol involves many washing and transfer steps that make it tedious to perform manually. With the VIAFLO 96, a handheld 96 channel electronic pipette, multistep protocols such as PCR clean-up and DNA purification can be performed quickly and efficiently, increasing throughput tremendously by transferring samples and reagents to all 96 wells at once. Thanks to its unique operating concept, the VIAFLO 96 remains as easy to use as a traditional handheld pipette and can even provide critical information (user-defined prompts) about the protocol steps.

    Key benefits

    • The VIAFLO 96 enables transfer of samples, reagents and wash solutions to 96 wells at once, increasing the throughput of magnetic bead-based DNA purification methods.
    • The partial tip loading of the VIAFLO 96 allows purification of fewer than 96 DNA samples if necessary; 8, 16, 24, 32, 40 or 48 GripTips can be loaded for easy purification of different numbers of samples.
    • The optimal immersion depth for removing supernatant or adding liquid right onto the samples is guaranteed by defining the z-height of the VIAFLO 96.
    • The Tip Align setting of the VIAFLO 96 automatically positions the tips in the center of the wells of a 96 well plate, avoiding any disturbance of the beads.

    Overview: How to automate PCR purification steps with VIAFLO 96 

    The VIAFLO 96 handheld electronic pipette with a three position stage (Plate holder options for VIAFLO 96/384) is used to purify DNA with AMPure XP beads from Beckman Coulter. The following protocol is an example of a set-up for 96 samples, where each well of a 96 well plate is filled with 10 μl of DNA sample and 18 μl of AMPure XP beads, then further processed with the VIAFLO 96. The PCR purification can be performed manually or semi-automated using the VIAFLO 96 in automatic mode. Custom-made VIALINK programs are provided. The VIALINK programs are set up according to the manufacturer’s protocol (AMPure XP Beckman Coulter).

    Downloads: Protocols for automated PCR with VIAFLO 96/384

    Here you can download the complete protocols used in this application and use them on your VIAFLO 96/384. The protocol can be customized with the VIALINK software.

    Step-by-step procedure

    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.

    Transfer AMPure XP beads from the stock solution into 12 PCR tubes

    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.

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

    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.

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

    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.

    Separating the magnetic beads from the PCR samples.

    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.

    Wash the magnetic beads twice with 70 % ethanol.

    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.

    Elute samples from the magnetic beads

    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.

    Transfer the sample eluates into the new 96 well plate.

    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.

    Automatic mode and partial tip load.

    Conclusion

    • The VIAFLO 96 is perfectly suited to magnetic bead purification in a 96 well format. An entire plate with 96 samples can be purified in a fraction of the time it would take with a traditional pipette.
    • Optimized tip immersion and pipette settings in combination with the use of Low Retention GripTips allow maximum sample recovery at the end of the purification protocol.
    • The VIAFLO 96 can guide the user through the entire protocol step by step, ensuring the correct workflow and enhancing the reproducibility of results.
    • The optional automatic mode of the VIAFLO 96 enables the instrument to operate on its own to minimize pipetting errors, making it even more ideal for use under a laminar flow cabinet.

    April 2020, by

    • Lydiane Saucède, PhD

      Application Scientist

      Email
    • Jan-Peter Baldin, PhD

      Product Manager

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    Any questions? I'm happy to help!

    • Tom Bentivegna

      Product Manager

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    Used instruments and accessories

    VIAFLO 96/384, Handheld Electronic Pipette Three position stage for 96 and 384 well plates 96 Channel Pipetting Head, 125 µl VIAFLO single channel, 1250 µl, electronic pipette VIAFLO 12 channel, 50 µl, electronic pipette PCR 96 well cooling block GripTip, 125 µl, Sterile, Filter, Low retention Automation Friendly Reagent Reservoir, 300ml BECKMAN COULTER Life Sciences: Agencourt AMPure XP ALPAQUA Accelerating Genomic Discovery: 96S Super Magnet Plate Thermo Scientific: Armadillo PCR Plate, 96-well, clear, clear wells CORNING: Corning® Thermowell ® GOLD 0.2 ml 8-Well PCR tube strips

    VIAFLO 96/384, Handheld Electronic Pipette

    With this 24, 96 and 384 channel pipette, INTEGRA meets the need for an affordable, compact and easy to use pipette that provides increased productivity. The unique operating concept makes VIAFLO 96/384 as easy to use as any traditional handheld pipette.

    Part No. 6001 / 6031

    See how it works
    The VIAFLO 96/384 is handheld, electronic pipette

    Three position stage for 96 and 384 well plates

    This accessory expands available stage positions for microplates, reservoirs and tips on a VIAFLO 96 or VIAFLO 384 from 2 to 3 thereby enhancing workflow in a wide range of applications. Beneficially the Three Position Stage also features an indexing function to allow access to 384-well plates using a 96-channel pipetting head.

    Part No. 6230

    See all plate holder options
    Three position stage for 96 and 384 channel pipetting

    96 Channel Pipetting Head, 125 µl

    Users can simply change the pipetting head of their VIAFLO 96 and VIAFLO 384 to guarantee optimal matching of the available volume range to the application performed. Changing a pipetting head of VIAFLO 96 and VIAFLO 384 is easy and takes less than one minute.

    Part No. 6102

    Find out more about VIAFLO 96/384 Pipetting Heads Specifications
    VIAFLO 96/384 96 channel pipetting head

    VIAFLO single channel, 1250 µl, electronic pipette

    VIAFLO pipettes feature 10 preset pipetting programs, for which only a few parameters such as volume and speed need to be defined.

    For more elaborate pipetting protocols, up to 40 user defined custom programs can be created. The custom program is a step-based setup queuing liquid handling steps such as aspirate, dispense, mix and more.

    Part No. 4014

    Visit product page
    INTEGRA’s range of VIAFLO electronic pipettes.

    VIAFLO 12 channel, 50 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. 4636

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

    PCR 96 well cooling block

    Aluminium 96 and 384 well PCR cooling blocks that keep reagents chilled during sample preparation in a PCR plate. The cooling blocks feature a SBS footprint.

    Part No. 6250

    Find more labware accessories
    PCR cooling block

    GripTip, 125 µl, Sterile, Filter, Low retention

    INTEGRA uses a unique polypropylene blend to mold Low Retention GripTips with heightened hydrophobic properties. Using Low Retention GripTips prevents low surface tension samples from spreading out and “wetting” the inner wall of the tips, allowing them to bead-up for a maximum liquid recovery.

    Part No. 6565

    Learn more about low retention pipette tips
     Low Retention GripTips are offered in 6000 series racks as non-sterile, sterile and filter options.

    Automation Friendly Reagent Reservoir, 300ml

    INTEGRA offers two types of automation friendly reagent reservoirs:

    • 150 ml and 300 ml Clear Advantage™ reagent reservoirs which are made of both crystal clear virgin polystyrene and polypropylene. Both options fit into a reusable automation friendly base with bold, crisp, clearly visible graduation markings.
       
    • The 300 ml open well, 12 column and 8 row polypropylene reservoir blocks offer improved chemical compatibility.
    Find out more about Automation Friendly Reagent Reservoirs
    300 ml Reservoir filled

    BECKMAN COULTER Life Sciences: Agencourt AMPure XP

    A highly efficient, easily automated PCR purification system that delivers superior quality DNA with no salt carryover. Requiring no centrifugation or filtration, Agencourt AMPure XP can be easily used in manual and automated 96- or 384-well formats.

    Source: Website Beckman Coulter

    Visit supplier website

    ALPAQUA Accelerating Genomic Discovery: 96S Super Magnet Plate

    SBS-compliant ring magnet plate with extra strong ring magnets ideal for large volume separations. Compatible with many 96-well PCR plates, round bottom deep well and half deep well plates.

    Source: Website Alpaqua Engineering

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    Thermo Scientific: Armadillo PCR Plate, 96-well, clear, clear wells

    The Thermo Scientific Armadillo PCR plates are the ultimate in high performance PCR plastics. Combining the rigidity of a polycarbonate frame with thin-walled polypropylene wells, the Armadillo plate provides superior thermal cycling performance under all conditions without warping. These 96-well plates are clear with clear wells.

    Source: Website Thermo Fisher

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    CORNING: Corning® Thermowell ® GOLD 0.2 ml 8-Well PCR tube strips

    • Tube strips consist of eight 0.2 mL tubes connected together
    • Cap strips are sold separately
    • Made of thin wall polypropylene and designed for precise fit in heat blocks for optimum heat transfer
    • Certified DNase- and RNase-free
    • Autoclavable at 121°C

    Source: Website Merck KGaA

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    Customer’s voice

    We've had two VIAFLO 96's for about 2 years now. We've been using them on our newest assays, one of which has multiple reagent additions and requires a liquid-liquid extraction. The VIAFLO's make this very easy. We haven't had any issues with them really. We also get the yearly PM plan which makes sure our heads stay calibrated and all is working properly. One of the service engineers gave me a tutorial on how to set up programs and it's very easy to do!

    Sarah Goff, New England Newborn Screening