Efficient and automated 384 well qPCR set-up with the ASSIST PLUS pipetting robot

  • Efficient and automated 384 well qPCR set-up with the ASSIST PLUS pipetting robot

    Using the ASSIST PLUS pipetting robot to automate set-up for a 384 well plate qPCR.

    Setting up a qPCR is a tedious process consisting of multiple pipetting steps. One particularly challenging task is reformatting from microcentrifuge tubes into a 384 well plate, which is time consuming and requires a lot of concentration. Another common problem is the loss of valuable and expensive substances, such as Mastermix and precious samples, due to the reservoir dead volume. The ASSIST PLUS pipetting robot, in combination with the VIAFLO and VOYAGER electronic pipettes, streamlines the workflow and increases the throughput and the reproducibility of qPCR set-ups, with minimal manual input. The loss of expensive substances or valuable samples due to reformatting errors is eliminated. The unique design of the ASSIST PLUS pipetting robot, together with the intuitive VIALAB software, offers exceptional flexibility and straightforward implementation.

  • Table of contents

    Setting up a qPCR is a tedious process consisting of multiple pipetting steps. One particularly challenging task is reformatting from microcentrifuge tubes into a 384 well plate, which is time consuming and requires a lot of concentration. Another common problem is the loss of valuable and expensive substances, such as Mastermix and precious samples, due to the reservoir dead volume. The ASSIST PLUS pipetting robot, in combination with the VIAFLO and VOYAGER electronic pipettes, streamlines the workflow and increases the throughput and the reproducibility of qPCR set-ups, with minimal manual input. The loss of expensive substances or valuable samples due to reformatting errors is eliminated. The unique design of the ASSIST PLUS pipetting robot, together with the intuitive VIALAB software, offers exceptional flexibility and straightforward implementation.

Key benefits

  • Automating the qPCR set-up with the VIAFLO 16 channel electronic pipette and the ASSIST PLUS pipetting robot allows considerably faster sample preparation, freeing up time for scientists to focus on other experiments.
  • Automation of VOYAGER adjustable tip spacing pipettes with the ASSIST PLUS offers a reliable pipetting method that requires minimal manual intervention and eliminates the risk of reformatting errors.
  • The use of Low Retention GripTips with heightened hydrophobic properties and SureFlo™ low dead volume reservoirs with an anti-sealing array helps to save precious samples and Mastermix. Combined with the high pipetting accuracy and precision of the ASSIST PLUS pipetting robot, this enables exceptionally low dead volumes to be achieved.
  • The ASSIST PLUS pipetting robot, in combination with the intuitive VIALAB software, is quick to set up and easy to use.

Overview: qPCR set-up

The ASSIST PLUS pipetting robot is used to set up a 384 well format qPCR by pipetting 64 samples in triplicate with two different Mastermixes for the detection of two genes of interest (GOI 1 and GOI 2).
The protocol is divided into two programs that guide the user through all the steps of the qPCR set-up:

  • Program 1: Mastermix_qPCR
  • Program 2: Samples_qPCR

The ASSIST PLUS pipetting robot operates a VIAFLO 16 channel 125 μl electronic pipette with 125 μl Sterile, Filter, Low Retention GripTips for Program 1 and a VOYAGER 8 channel 12.5 μl electronic pipette with 12.5 μl Sterile, Filter, Low Retention GripTips for Program 2.

Downloads: Protocols and reports for automated qPCR set-up 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: Program 1 - Mastermix transfer (Mastermix_qPCR)

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

Deck position A:

Dual reservoir adapter – 2 x 10 ml reagent reservoir with SureFlo anti-sealing array (Figure 2) containing Mastermix 1 and 2.

Deck position B:

384 well PCR plate, placed on an INTEGRA cooling block in the landscape position.

Figure 1: Set-up for the Mastermix transfer.
Figure 1: Set-up for the Mastermix transfer. Position A: Dual reservoir adapter with 2 x 10 ml reagent reservoirs with SureFlo anti-sealing array. Position B: 384 well PCR plate, placed on an INTEGRA cooling block.
The INTEGRA dual reservoir adapter accommodates both 10 ml reagent reservoirs on one deck position.
Figure 2: The INTEGRA dual reservoir adapter accommodates both 10 ml reagent reservoirs on one deck position.

Step-by-step procedure

1. Transfer Mastermixes into the 384 well plate

Add Mastermixes 1 and 2 into the left and right sides of the 384 well PCR plate, respectively.

Use an EVOLVE 5000 μl manual pipette with 5000 μl Sterile, Filter, Low Retention GripTips to fill the left 10 ml reagent reservoir with SureFlo anti-sealing array with 1.6 ml of Mastermix 1 and the right reservoir with 1.6 ml of Mastermix 2 (Position A). Select and run the VIALAB program ‘Mastermix_qPCR’ on the VIAFLO 16 channel 125 μl electronic pipette with 125 μl Sterile, Filter, Low Retention GripTips. The ASSIST PLUS pipetting robot automatically transfers 7.5 μl of Mastermix 1 (pink) into the left half of the 384 well PCR plate and 7.5 μl of Mastermix 2 (blue) into the right half (Figure 3) using the Repeat Dispense mode with a tip touch on the surface of the liquid to increase pipetting precision. Figure 4 shows the pipetting robot transferring the Mastermix into a 384 well plate.

    Figure 3: Pipetting scheme for Mastermixes 1 (pink) and 2 (blue).
    Figure 3: Pipetting scheme for Mastermixes 1 (pink) and 2 (blue).

    Tips:

    • A pre- and post-dispense step is recommended to increase the accuracy and precision of pipetting. The pre- and post-dispense volume should be between 3 and 5 % of the nominal volume of the pipette.
    • The Low Retention GripTips are made from a unique polypropylene blend with heightened hydrophobic properties for superior accuracy and precision while pipetting viscous and low surface tension liquids.
    • The reservoirs’ SureFlo anti-sealing array and a unique surface treatment that spreads liquid evenly enable the pipette tips to sit on the bottom and still aspirate liquids accurately, reducing dead volumes.
    Example of the ASSIST PLUS pipetting robot transferring a Mastermix into a 384 well PCR plate.
    Figure 4: Example of the ASSIST PLUS pipetting robot transferring a Mastermix into a 384 well PCR plate.

    Experimental set-up: Program 2 - Sample transfer (Samples_qPCR)

    Prepare the pipetting robot deck as follows (Figure 5):

    Deck position B:

    384 well PCR plate, placed on an INTEGRA cooling block.

    Deck position C:

    INTEGRA 1.5 ml microcentrifuge tube rack, with tubes containing samples 1-32.

    Set-up for the sample transfer protocol.
    Figure 5: Set-up for the sample transfer protocol. Position B: 384 well PCR plate, placed on an INTEGRA cooling block. Position C: INTEGRA 1.5 ml microcentrifuge tube rack, with tubes containing samples 1-32 (Figure 6).
    Example of the ASSIST PLUS pipetting samples from the INTEGRA microcentrifuge tube rack into a 96 well plate.
    Figure 6: Example of the ASSIST PLUS pipetting samples from the INTEGRA microcentrifuge tube rack into a 96 well plate.

    Step-by-step procedure

    1. Sample transfer into the 384 well plate

    Add the 64 samples in triplicate to the Mastermixes.

    Place samples 1-32 in an INTEGRA 1.5 ml microcentrifuge tube rack on Position C. Run the VIALAB program ‘Samples_qPCR’ on a VOYAGER 8 channel 12.5 μl electronic pipette to start the sample transfer. The ASSIST PLUS transfers 2.5 μl of the first 32 samples in triplicate into Mastermixes 1 and 2 (Figure 7, yellow/brown), using the Repeat Dispense mode with a tip touch on the side of the well to make sure that no droplets adhere to the GripTips. After this step, a prompt informs the user to place the second series of samples (33-64) on Position C. The ASSIST PLUS pipetting robot continues by transferring 2.5 μl of the samples in triplicate into the other half of Mastermixes 1 and 2 (Figure 7, green).

    Tip: Use Sterile, Filter, Low Retention GripTips for optimal liquid recovery of precious solutions, such as Mastermix and samples.

    ASSIST_PLUS_qPCR_V00 4/5 Application Note Figure 6: Example of the ASSIST PLUS pipetting samples from the INTEGRA microcentrifuge tube rack into a 96 well plate. Figure 7: Pipetting scheme of the qPCR assay.
    Figure 7: Pipetting scheme of the qPCR assay.

    Remarks

    VIALAB software:

    The VIALAB program can easily be adapted to fit the user’s demands, especially if specific labware, incubation times or protocols are needed.

    Partial plates:

    The 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 time required for a 384 well qPCR set-up can be reduced from 1.5 hours using a single channel pipette to 12 minutes using the ASSIST PLUS pipetting robot in combination with VIAFLO 16 channel and VOYAGER 8 channel pipettes.
    • The ASSIST PLUS, together with the VOYAGER adjustable tip spacing pipette, guarantees perfectly reproducible test results and eliminates all risks of reformatting errors when transferring samples from microcentrifuge tubes into a 384 well plate.
    • INTEGRA’s Low Retention GripTips increase pipetting precision for viscous or low surface tension liquids. The reagent reservoirs with SureFlo anti-sealing array reduce the dead volume of costly reagents and precious samples.
    • The intuitive VIALAB qPCR program is quick to set up and easy to use or adapt to other pipetting protocols.

    Any questions? I'm happy to help!

    • Jan-Peter Baldin, PhD

      Product Manager

      Email

    Used instruments and accessories

    ASSIST PLUS, Pipetting Robot Communication module for INTEGRA electronic pipettes Rack for 1.5/2 ml microcentrifuge tubes Dual Reservoir Adapter PCR 384 well cooling block VOYAGER, 8 Channel, 12.5 µl VIAFLO 16 channel, 125 µl, electronic pipette EVOLVE manual pipette 5000 µl 10 ml, Disposable Reservoirs, Sterile, SureFlo™ anti-sealing array, PS GripTip, 12.5 µl, Sterile, Filter, Low Retention GripTip, 125 µl, Sterile, Filter, Low retention GripTip, 1250 µl, Sterile, Filter, Low Retention Greiner Bio-One: 384 Well Polypropylene Sapphire Microplates

    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

    Visit product page
    ASSIST PLUS Pipetting Robot

    Communication module for INTEGRA electronic pipettes

    For pairing INTEGRA electronic pipettes with the ASSIST PLUS.

    Part-Nr. 4221

    Find out more about pipette accessories

    Rack for 1.5/2 ml microcentrifuge tubes

    Hold up to 48 tubes on removable carriers for easy loading and storage.

    Part No. 4540

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    Rack for 1.5/2 ml microcentrifuge tubes

    Dual Reservoir Adapter

    The dual reservoir adapter makes this system ideal for assays that required sequential addition of several low volume reagent mixes. This adapter holds two 10 ml, 25 ml or divided reagent reservoirs side by side in an aluminum frame, and can be pre-chilled to keep reagents cool during processing.

    Part No. 4547

    Find out more about ASSIST PLUS accessories
    Dual reservoir adapter with 2 didived reservoirs

    PCR 384 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. 6255

    Find more labware accessories
    384-well PCR cooling plate

    VOYAGER, 8 Channel, 12.5 µl

    VOYAGER pipettes allow the tip spacing to expand anywhere between 4.5 mm and 33 mm at the push of a button.

    • Single handed operation leaves the other hand free to handle labware.
    • On the fly access to up to 3 user-defined tip spacings.
    • These user defined tip spacings are saved. No need to memorize spacings of different labware formats.

    Part No. 4721

    See it work
    VOYAGER adjustable tip spacing pipette

    VIAFLO 16 channel, 125 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. 4642

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

    EVOLVE manual pipette 5000 µl

    Traditional pipettes utilize a single rotating plunger to set the volumes. This approach makes frequent adjustment of volumes tedious, especially when switching from lower to higher volumes. The EVOLVE manual pipette has been designed to overcome this pain point and offers many other advantages:

    • Quick set dials
      Adjust volumes more than ten times faster thanks to three adjustable dials.
    • Reduced risk for repetitive strain injuries
      Two differently sized plunger springs allow users to customize the force needed to depress the plunger to their preference.
    • Best in class pipette tips
      GripTips are always firmly attached, perfectly aligned and feature a low attachment and ejection force.
    • Wide product range
      EVOLVE pipettes are available for volumes from 0.2 µl to 5000 µl, as single, 8, 12 and 16 channel models.

    Part No. 3019

    See it work
    EVOLVE single channel manual pipette.

    10 ml, Disposable Reservoirs, Sterile, SureFlo™ anti-sealing array, PS

    SureFlo™ anti-sealing array prevents pipette tip seal off which can cause liquid to pop into the tip, filter or even the pipette itself.

    Part No. 4371

    Find out more about Multichannel Reagent Reservoirs
    Reagent reservoirs 10ml, 25ml, 100ml

    GripTip, 12.5 µl, Sterile, Filter, Low Retention

    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. 6555

    Learn more about GripTips pipette tips for Benchtop Pipetting Systems
    Integra-Relaunch_Website-Output-Produktebilder-Product_Overview-GripTips.png

    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.

    GripTip, 1250 µl, Sterile, Filter, Low Retention

    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. 6545

    Learn more about GripTips pipette tips for Benchtop Pipetting Systems
    Integra-Relaunch_Website-Output-Produktebilder-Product_Overview-GripTips.png

    Greiner Bio-One: 384 Well Polypropylene Sapphire Microplates

    SAPPHIRE MICROPLATE, 384 WELL, PP, PCR, WITH SKIRT, NATURAL, 15 PCS./BAG

    Source: Website Greiner Bio One

    Visit supplier website

    Customer’s voice

    You must try this. You will fall in love with it. I am so grateful to have had the opportunity to use this tool. It's really more than awesome. I love many products from Integra, which upgrade the quality of our research enormously. Just be ready to enjoy every step of your research with Integra. (Review published on Select Science)

    Grace Lee, University of Utah School of Medicine