Soil DNA was extracted using the Norgen’s Soil DNA Isolation (Magnetic Bead System) kit (Norgen Biotek Corp., Ontario, Canada). Section 2 (2.3 DNA isolation using Manual Method) of the protocol (DNA isolation from the prepared soil lysate) was performed in Opentrons OT-2 (Opentrons Labworks, Inc., UK). The protocol was designed using Opentrons Protocol Designer Version 8.4.4 and divided into three sub-protocols to prevent errors. In the effort to save time and enhance precision, mixing steps were done manually. The OT-2 made scripts can be found at this repository.
Protocol for only one plate well column filled (8 wells) available in standard-protocol branch
The protocol was divided into three to minimize errors with the tip racks counted in the script. Every labware used was DNA-free. Every mixture of buffers and its addition to reservoirs was freshly made. Specifications:
- Pipette: Right mount P300 8-Channel GEN2
- Opentrons OT-2 96 Filter Tip Rack 200 μL
- Modules used: Magnetic Module GEN2 and Heater-Shaker Module GEN1 with Opentrons 96 Deep Well Heater-Shaker Adapter
- Labware: NEST 1 Well Reservoir 195 mL (for discharge);
- NEST 12 Well Reservoir 15 mL (to place buffers) and NEST 96 Well Plate 100 μL PCR Full Skirt (final DNA extracts)
Protocol 1 Addition of magnetic beads and first and second wash with Solution WN
Mix of magnetic beads:
Per column of a 15mL NEST 12 well reservoir. To the amount set by the kit manufacturer, it was multiplied by eight wells (number of wells per column in a 96-well plate) and by 15% extra volume for pipetting errors. To avoid potential cross-contamination between columns, the full volume of the mixture (5828 μL) was added to each 12-well plate, rather than reducing the number of wells used to six.
- Binding Buffer B: 300 x 8 x 0.15 = 2760 μL
- 96% EtOH: 320 x 8 x 0.15 = 2944 μL
- Magnetic beads: 20 x 8 x 0.15 = 184 μL
- Total: 5828 μL
Solution WN:
Per column of a 15mL NEST 12 well reservoir. To the amount set by the kit manufacturer, it was multiplied by eight wells (number of wells per column in a 96-well plate) and by 15% extra volume for pipetting errors. 1000 μL x 8 x 0.15= 8800 μL Two full reservoirs with 8800 μL in each well.
Protocol 2: Addition of first and second wash 80% EtOH
Per column of a 15mL NEST 12 well reservoir. To the amount set by the kit manufacturer, it was multiplied by eight wells (number of wells per column in a 96-well plate) and by 15% extra volume for pipetting errors.
- 1000 μL x 8 x 0.15= 8800 μL
- Two full reservoirs with 8800 μL in each well.
Protocol 3: Elution of DNA from the magnetic beads
Per column of a 15mL NEST 12 well reservoir. To the amount set by the kit manufacturer, it was multiplied by eight wells (number of wells per column in a 96-well plate) and by 15% extra volume for pipetting errors.
- 50 μL x 8 x 0.15= 5520 μL
- One well is filled with 5520 μL in a single reservoir.