Automated qPCR master mix preparation with the D-ONE single channel pipetting module and the ASSIST PLUS pipetting robot

  • Automated qPCR master mix preparation with the D-ONE single channel pipetting module and the ASSIST PLUS pipetting robot

    qPCR master mix preparation

    Automating qPCR master mix preparation is often considered extremely tedious, due to the need to constantly change program set-up based on varying master mix volumes and sample numbers. However, the D-ONE single channel pipetting module for the ASSIST PLUS pipetting robot simplifies the programming of automated qPCR master mix preparation protocols down to a single step. The D-ONE individually reaches tubes of different qPCR master mix components, while the VIALAB program takes care of all calculations and provides automatic tip selection.

     

    This solution allows master mixes to be pipetted into any tube or reservoir, enabling large-scale qPCR master mix preparation, with subsequent distribution to plates with multichannel pipettes to speed up the process. Here, we verify a SYBR® Green qPCR protocol prepared directly in a divided reservoir using the D-ONE on the ASSIST PLUS.

  • Table of contents

    Automating qPCR master mix preparation is often considered extremely tedious, due to the need to constantly change program set-up based on varying master mix volumes and sample numbers. However, the D-ONE single channel pipetting module for the ASSIST PLUS pipetting robot simplifies the programming of automated qPCR master mix preparation protocols down to a single step. The D-ONE individually reaches tubes of different qPCR master mix components, while the VIALAB program takes care of all calculations and provides automatic tip selection.

     

    This solution allows master mixes to be pipetted into any tube or reservoir, enabling large-scale qPCR master mix preparation, with subsequent distribution to plates with multichannel pipettes to speed up the process. Here, we verify a SYBR® Green qPCR protocol prepared directly in a divided reservoir using the D-ONE on the ASSIST PLUS.

Key benefits

  • Fully automated liquid handling of various qPCR master mix components on the ASSIST PLUS pipetting robot with the D-ONE ensures the reproducibility of results.
  • qPCR master mix components and volumes can be easily adjusted in VIALAB to perform different qPCR protocols. The final calculation is automatically done by VIALAB according to the selected reaction count.
  • The D-ONE's liquid level detection allows the use of reagents with different aliquot volumes, and informs the user if there is insufficient liquid. Furthermore, the automated tip selection guarantees high precision when pipetting different volumes.
  • qPCR master mixes can be pipetted into various target vessels, including INTEGRA's multichannel reservoirs with a very low dead volume, to speeds up the plating of master mixes using a multichannel pipette.

Downloads: App note and protocol for automated qPCR master mix preparation with the D‑ONE single channel pipetting module and the ASSIST PLUS pipetting robot

Here you can download the complete protocol used in this application and use them on your D‑ONE single channel pipetting module and the ASSIST PLUS pipetting robot. The protocol can be customized with the VIALAB software.

Overview: How to automate protocols for SYBR Green qPCR master mix preparation

 

Experimental set-up

The 5-1250 µl D-ONE single channel pipetting module with 125 µl and 1250 µl sterile, filter, GRIPTIPS® is mounted on the ASSIST PLUS pipetting robot.

An INTEGRA divided polypropylene reagent reservoir with SureFlo™ anti-sealing array is placed onto the dual reservoir adapter on deck position A. An INTEGRA tube rack with nuclease-free water (A1), SYBR Green master mix reagent (B1), forward primer (C1) and reverse primer (D1) is placed on position B (Figure 1).

Graphical representation of the ASSIST PLUS pipetting robot deck set-up with INTEGRA dual adapter for divided reagent reservoirs, an INTEGRA tube rack with qPCR master mix components, and the D-ONE module.
Figure 1: Deck set-up for qPCR master mix preparation in divided reagent reservoir. Position A: Target – INTEGRA dual reservoir adapter with divided reagent reservoir (blue). Position B: Source – INTEGRA tube rack for 2 ml tubes with screw caps and 1.5 ml microcentrifuge tubes (pink). Position C: empty.

Step-by-step procedure

1. Calculation of qPCR master mix components

Adjusting the number of reactions in VIALAB.

The reaction count is predefined to 98 reactions to fill a full 96 well qPCR plate (9 µl reaction volume, 10 µl dead volume). The user defines the reagent ID and its volume per reaction in VIALAB, which can be changed according to the specific qPCR protocol. By adjusting the reaction count, the transfer volume of all qPCR master mix components is calculated automatically, as well as selection of the appropriate GRIPTIP (Figure 2).

Graphical representation of the VIALAB qPCR master mix preparation step with four predefined qPCR master mix components, their volumes, and the total reaction count.
Figure 2: In the master mix step, VIALAB calculates the volume of qPCR master mix components according to the reaction count.

Tips:

  • Once a program with a master mix step is set up, it can be reopened and only the number of reactions needs to be changed prior to the start of the run. No reprogramming is needed, saving time for the operator.
  • Depending on the final volume calculated by VIALAB, the tip type (125/1250 µl) is automatically chosen, further alleviating the need for user input.

 

2. Transfer of qPCR master mix components

The different components of qPCR master mix are added one by one.

Select and run the VIALAB program ‘Master_mix_prep_in_reservoir’. The 5-1250 µl D-ONE pipetting module with 1250 µl sterile, filter GRIPTIPS aspirates 294 µl of nuclease-free water from a 2 ml tube (Position B - A1), and dispenses it into the 5 ml compartment of the divided reservoir, Position A - A1. The D-ONE module then changes tips automatically between different reagents, and transfers 490 µl of SYBR Green master mix from B1 to A1 of the divided reservoir. With 12.5 µl sterile, filter GRIPTIPS, the D-ONE then transfers 49 µl each of the forward primer (C1) and reverse primer (D1) into A1 of the divided reservoir. With a new 1250 µl sterile, filter GRIPTIP, the D-ONE mixes 600 µl of SYBR Green master mix three times, in three different offsets within the 5 ml compartment of the divided reservoir -A1.

Tip:

  • Different mixing offsets in the reagent reservoir improve the homogeneity of the qPCR master mix components. Mixing volume can be adjusted easily when reducing the reaction count, while mixing cycles can be increased to produce even higher volumes of qPCR master mix.

 

3. Results

We used a SYBR Green qPCR protocol to amplify a 250 bp fragment from the fifth variable region of bacterial 16S rRNA gene with the QuantStudio™ 3 (Thermo Fisher Scientific) to demonstrate proper homogeneity when performing qPCR master mix preparation in divided reagent reservoirs. The master mix was prepared both manually in tubes and with the ASSIST PLUS pipetting robot and the D-ONE single channel pipetting module in the divided reservoir.

Figure 3 represents the amplification of a section of six replicates from a qPCR plate using SYBR Green master mix produced with the ASSIST PLUS and the D-ONE in a divided reservoir (green), compared to six replicates using master mix produced manually in tubes (orange). Both qPCR master mix preparations show valid amplification, demonstrating the proper homogeneity of qPCR master mix components.

Graphical representation of the SYBR Green qPCR amplification plot showing the transfer of master mix from tubes and divided reservoirs.
Figure 3: Amplification plot of a 250 bp fragment of the fifth variable region of bacterial 16S rRNA showing a section of six replicates.

Remarks

  • VIALAB software: VIALAB programs can be easily adapted to your specific labware and protocols, such as when partial plates are needed.
  • Partial plates: Programs can be adapted at any time to varying sample numbers, giving laboratories total flexibility to meet current and future demands.

Conclusion

  • The ASSIST PLUS pipetting robot and D-ONE single channel pipetting tool, together with the VIALAB master mix step enables the easy and reliable automation of master mix preparation, including automatic volume calculations, tip selection and liquid level detection.
  • The ASSIST PLUS pipetting robot is the ideal solution for automating qPCR master mix preparation, set-up, and sample addition. It maximizes hands-free time and increases reproducibility by eliminating any variability introduced by operators.
  • With its small footprint, the ASSIST PLUS fits into biosafety cabinets to ensure sterile conditions while pipetting precious qPCR master mix components.
  • The D-ONE single channel pipetting module can individually transfer various qPCR master mix components, and effectively mix them in divided reservoirs for large scale qPCR master mix preparation.
  • The homogeneity of qPCR master mix components can be guaranteed by adjusting mixing volumes and cycles, setting different mixing offsets, and the unique surface treatment of the SureFlo polypropylene divided reagent reservoir.
  • The divided reagent reservoir can then be easily accessed using an 8 channel 12.5 µl VOYAGER adjustable tip spacing pipette to quickly set up qPCR plates.

Ask our expert. Leave a comment!

Write us if you have any questions regarding the application note or one of our instruments.

Any questions? I'm happy to help!

  • Rebecca Bodmer

    Application Specialist

    Email

Instruments and accessories

ASSIST PLUS, Pipetting Robot D-ONE Single Channel Pipetting Module, 0.5-300 µl Tip deck for D-ONE on ASSIST PLUS Rack for 1.5/2 ml microcentrifuge tubes Dual Reservoir Adapter Divided Reagent Reservoir, Sterile, SureFlo anti-sealing array, 25 ml, PP GRIPTIPS®, 125 µl, Sterile, Filter (for automation systems) GRIPTIPS®, 1250 µl, Sterile

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

D-ONE Single Channel Pipetting Module, 0.5-300 µl

The D-ONE single channel pipetting module offers hands-free transfers from individual tubes or wells on the ASSIST PLUS pipetting robot. This makes it the perfect add-on for labs that want to go beyond the possibilities of multichannel pipetting to access unlimited applications. It is designed to offer easy automation of normalization, hit picking and master mix preparation, freeing up your time while eliminating transcription errors.

Learn more about the D-ONE Single Channel Pipetting Module
D-ONE pipetting module 1ch 0.5-300ul

Tip deck for D-ONE on ASSIST PLUS

  • Supports accommodation of 1 x 12.5 µl/ 125 µl tip box and 1 x 300 µl/ 1250 µl tip box
  • Compatible with D-ONE Pipetting Module 0.5 - 300 µl and 5 - 1250 µl
Learn more about the Tip Deck
D-ONE tip deck

Rack for 1.5/2 ml microcentrifuge tubes

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

Part No. 4540

Show all labware accessories
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

Divided Reagent Reservoir, Sterile, SureFlo anti-sealing array, 25 ml, PP

Lowest Dead Volume On The Market!

25 ml divided reservoirs are comprised of 5 and 10 ml compartments, while still nesting comfortably in the standard 25 ml reusable, sturdy base. 
Each reservoir features the SureFlo™ anti-sealing array and hydrophilic surface treatment providing especially low dead volumes.

Part No. 4356 / 4357

Find out more about Divided Reagent Reservoirs
divided-reagent-reservoir-to-well-plate.jpg

GRIPTIPS®, 125 µl, Sterile, Filter (for automation systems)

GRIPTIPS® 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. 6465

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

GRIPTIPS®, 1250 µl, Sterile

GRIPTIPS® 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
1250 µl, Non-sterile, GripTips

Customer’s voice

Implementing this liquid handler in our molecular workflow has cut production time dramatically with the ease and reproducibility this instrument offers. Prep can be done in half the time and supports new lab personnel carry out multiple steps in our workflow with accuracy and confidence. The flexibility of the unit is also incredible with the ability to edit programs in real time or on the pc, reassign pipettes to base units freely as workflows requires. This liquid handler is a staple in our laboratory

Gentech Diagnostics, CAITLIN OSBORN