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, with 125 and 1250 μl sterile, filter GRIPTIPS® pipette tips, is mounted on the ASSIST PLUS. 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).

Step-by-step procedure

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 the selection of the appropriate GRIPTIPS (Figure 2).

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.

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, 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 then changes tips automatically between different reagents, and transfers 490 μl of SYBR Green master mix from Position B – B1 to Position A – A1. With 12.5 μl sterile, filter GRIPTIPS, the D-ONE then transfers 49 μl each of the forward primer (Position B – C1) and reverse primer (Position B – D1) into the divided reservoir (Position A – A1). With new 1250 μl sterile, filter GRIPTIPS, the D-ONE mixes 600 μl of SYBR Green master mix 3 times, in 3 different offsets within the 5 ml compartment of the divided reservoir (Position A – A1).

Tip:

• Different mixing offsets in the reagent reservoir improve the homogeneity of the qPCR master mix components. The 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.

Results

A SYBR Green qPCR protocol was used to amplify a 250 bp fragment from the fifth variable region of the bacterial 16S rRNA gene. A QuantStudio™ Real-Time PCR System 3 (Thermo Fisher Scientific) was used 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 and the D-ONE in the divided reservoir.

Figure 3 represents the amplification of a section of 6 replicates from a qPCR plate using SYBR Green master mix prepared with the ASSIST PLUS and the D-ONE in a divided reservoir (green), compared to 6 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.

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.