Sep 25, 2023
Opentrons Pipeline: DNA Extraction with the Omega Biotek Stool Kit
- 1San Diego Zoo Wildlife Alliance;
- 2Field Projects International
- In Situ LaboratoriesTech. support email: [email protected]
Protocol Citation: Kristina Vsevolodova, Gideon Erkenswick, Mrinalini Watsa 2023. Opentrons Pipeline: DNA Extraction with the Omega Biotek Stool Kit . protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp39njvzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 19, 2023
Last Modified: September 25, 2023
Protocol Integer ID: 88037
Keywords: Opentrons, OT2, DNA extraction, Extraction, Omega Biotek, Stool , Stool extraction, insitulabs, automated, dna extraction with the omega biotek stool kit, omega biotek stool kit, opentrons pipeline, dna extraction, opentron, automated pipetting robot, pipetting robot, pipeline, automated pipeline, extraction, well plate of dna, stool lysate, omega biotek, 200ul opentron, dna, stool, robot, filtered tip box, lysing of the stool
Funders Acknowledgements:
Gordon and Betty Moore Foundation
Grant ID: 9772
Revive and Restore Catalyst Science Fund
Grant ID: 2021-024
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Abstract
This protocol is an automated pipeline to extract a full 96-well plate of DNA from stool lysates prepared in two separate plates. Lysing of the stool can be performed as you wish, depending on species and their diet. The protocol itself begins after lysates have been created.
This protocol was developed and optimized for the following:
Platform: Opentrons OT-2 automated pipetting robot
Kit: Omega Biotek
Tips Used: 4 boxes (4 x 200uL Opentrons Filtered Tip boxes)
Recommended number of samples: 96
Guidelines
This protocol is recommended for a full 96-well plate. Do not use it for less than 12 columns. A version for variable sample numbers is forthcoming in the next version.
Things to consider:
Before beginning: Load your lysates into two plates, a 1 mL deep well with 300uL of lysate and a 200uL Plate with 200uL of lysate. This is because, during the COVID pandemic, locating 2mL deep well plates for small labs (i.e. to buy in numbers less than 10,000) was impossible to do. So, we made our protocol flexible to be able to use a larger amount of lysate (total of 500uL) without the 2mL deepwell plate.
Step 4: The 50mL Falcon tubes are recommended as means of sterile transport of accurate reagent amounts between the lab and the OT-2. They can be replaced with a different brand of tube or reservoir.
Step 6: Import the labware file BEFORE you import your protocol or it will give an error. This protocol has been validated against Opentrons software app version 6.2.1
Step 7.2: In the newer software versions for the OT-2, calibration is not required before every run. In some cases the user will not be asked to calibrate the machine, and they should not have to.
Materials
- 2x Aluminium Seals
- 6x 2mL Tubes
- 100-1000µL pipette
- 1000µL pipette tips
- Incubator or water bath that can reach 70°C or more
Protocol materials
10% Bleach
Distilled Water
70% Alcohol
XP2 Binding BufferOmega BiotekCatalog #PDR040
Mag-Bind Particles CHOmega BiotekCatalog #MBPCH- 50
VHB BufferOmega BiotekCatalog #VHB-440
SPM BufferOmega BiotekCatalog #SPM-300
Elution BufferOmega BiotekCatalog #PDR048
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Qubit® 3.0 FluorometerThermo Fisher ScientificCatalog #Q33216
Quantus(TM) NGS Starter PackagePromegaCatalog #E5150
Quantus(TM) FluorometerPromegaCatalog #E6150
Quant-iT™ PicoGreen® dsDNA Assay KitLife TechnologiesCatalog #P11496
Nuclease-Free Water, 150mlPromegaCatalog #P1195
Mag-Bind Stool DNA 96 KitOmega BiotekCatalog #M4016-01
100% Ethanol
Before start
Clean the OT2 deck and walls with:
10% Bleach 1 rinse
Distilled Water 1 rinse
70% Alcohol 2 rinses
Note
Avoid wetting electronic parts.
Before starting
Ingredients
100% Ethanol
Nuclease-Free Water, 150mlPromegaCatalog #P1195
Mag-Bind Stool DNA 96 KitOmega BiotekCatalog #M4016-01 (Contains everything for extraction including lysis reagents)
OR separate reagents (not including lysis reagents):
- SPM BufferOmega BiotekCatalog #SPM-300
- VHB BufferOmega BiotekCatalog #VHB-440
- XP2 Binding BufferOmega BiotekCatalog #PDR040
- Mag-Bind Particles CHOmega BiotekCatalog #MBPCH- 50
- Elution BufferOmega BiotekCatalog #PDR048
Note
For QC on individual level theQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854 on a Qubit® 3.0 FluorometerThermo Fisher ScientificCatalog #Q33216 can be used
OR the Quantus(TM) NGS Starter PackagePromegaCatalog #E5150 on a Quantus(TM) FluorometerPromegaCatalog #E6150
For QC on plate level use theQuant-iT™ PicoGreen® dsDNA Assay KitLife TechnologiesCatalog #P11496
For the 1x96 Kit
Dilute SPM Buffer with 70 mL 100% Ethanol per bottle and store at Room temperature .
Dilute VHB Buffer with 28 mL 100% Ethanol per bottle and store at Room temperature .
For the 4x96 Kit
Dilute SPM Buffer with 70 mL 100% Ethanol per bottle and store at Room temperature .
Dilute VHB Buffer with 112 mL 100% Ethanol per bottle and store at Room temperature .
Materials
2x Aluminium Seals
6x 2mL Tubes
100-1000µL pipette
1000µL pipette tips
Incubator or water bath that can reach 70°C or more
Autoclave the NEST 1-Well Reservoirs, 195 mL and NEST 12-Well Reservoirs, 15 mL before use. These can be rinsed and autoclaved and reused and need not be purchased new for each extraction. Slight yellowing of product can occur, but we do not see that it affects final outcomes in any discernible way.
Opentrons Equipment List
Equipment
OT-2
NAME
Liquid handler
TYPE
Opentrons
BRAND
OT-2
SKU
On the right pipette mount use the P300M
Equipment
OT-2 8 Channel Electronic Pipette
NAME
Pipette
TYPE
Opentrons
BRAND
P300M
SKU
LINK
Magnetic Module to place in Slot 7
Equipment
OT-2 Magnetic Module GEN2
NAME
Module
TYPE
Opentrons
BRAND
999-00098
SKU
LINK
Prepare reagents
After the reagents are properly diluted and materials are ready, prepare the following amounts:
| A | B | C | D | |
| Item Name | Amount per sample [uL] | Amount for 96 samples [uL] | Amount for 96 samples * 1.1 overage[uL] | |
| Mag-bind Bead Particles CH Round 1 | 10 | 960 | 1056 | |
| XP2 Binding Buffer Round 1 | 300 | 28800 | 31680 | |
| Mag-bind Bead Particles CH Round 2 | 10 | 960 | 1056 | |
| XP2 Binding Buffer Round 2 | 300 | 28800 | 31680 | |
| Wash 1: VHB Buffer | 400 | 38400 | 42240 | |
| Wash 2: SPM Buffer | 400 | 38400 | 42240 | |
| Wash 3: SPM Buffer | 400 | 38400 | 42240 | |
| Elution Buffer | 100 | 9600 | 10560 |
Fill one Nest 50mL Falcon Tube with the amount of XP2 Binding BufferOmega BiotekCatalog #PDR040 Round 1 and Mag-Bind Particles CHOmega BiotekCatalog #MBPCH- 50 Round 1 required in the table, and mix well. Keep at Room temperature . Split between two tubes if needed.
Fill one Nest 50mL Falcon Tube with the amount of XP2 Binding BufferOmega BiotekCatalog #PDR040 Round 2 and Mag-Bind Particles CHOmega BiotekCatalog #MBPCH- 50 Round 2 required in the table, and mix well. Keep at Room temperature . Split between two tubes if needed.
Fill one Nest 50mL Falcon Tube with the volume indicated in the table with wash 1: VHB BufferOmega BiotekCatalog #VHB-440 . Keep at Room temperature .
Fill one Nest 50mL Falcon Tube with the volume indicated in the table with wash 2: SPM BufferOmega BiotekCatalog #SPM-300 . Keep at Room temperature .
Fill one Nest 50mL Falcon Tube with the volume indicated in the table with wash 3: SPM BufferOmega BiotekCatalog #SPM-300 . Keep at Room temperature .
Distribute Elution BufferOmega BiotekCatalog #PDR048 between each 2mL tube evenly.
Set an incubator or water bath to 70 °C and heatElution BufferOmega BiotekCatalog #PDR048 in the incubator to 70 °C .
OT-2 script definitions
Definition of samples and labware:
Lysed Sample Plate 2 200uL
Remainder of lysed samples that will be added to Slot 7 in second round of bead incubation.
Position: Slot 2, 96-Well PCR Plate Non-skirt, 200µl with 200uL of sample lysis on top of an empty Nest skirted PCR Plate (used as a base)
Name in the Deck: Lysis plate 2
Labware name in the protocol: denvillewithaxygenbase_96_wellplate_200ul
Sample name in the script: lysate
Lysed Sample Plate 1 300uL
Lysed samples that will undergo the first round of bead incubation.
Position: Slot 7, Opentrons Magnetic Module with VWR 96-Well Deep Well Plate full of 300uL of sample lysis on top
Name in the Deck: Lysis plate 1
Labware name in the protocol: vwr_96_wellplate_1000ul
Sample name in the script: magsamps
Sample Elution 1
Samples that have been eluted from the beads for the first round elution 1.
Name in the Deck: Sample Elution Plate 1
Labware name in the protocol: nest_96_wellplate_100ul_pcr_full_skirt
Sample name in the script: eluates
Sample Elution 2
Samples that have been eluted from the beads for the second round elution 2.
Position: Slot 2, 96-Well PCR Plate Non-skirt, 200µl with 200uL of sample lysis on top of an empty Nest skirted PCR Plate is replaced with new Nest skirted PCR Plate (to receive elution 2)
Name in the Deck: Sample Elution Plate 2
Labware name in the protocol: nest_96_wellplate_100ul_pcr_full_skirt
Sample name in the script: eluates2
Prepare the OT-2
Before loading your protocol, load the following labware files into your Opentrons app: 
denville_96_axygenbase_200ul.json25KB
denville_96_axygenbase_200ul.json25KB This labware definition allows us to use a non-skirted plate in the Opentrons app by inserting it into a skirted plate, and also allows us to use a 200uL plate (where our skirted plates that clip in are only 100uL. Feel free to replace with your own labware here).
vwr_96_wellplate_1000ul.json11KB This labware definition is for the 1mL deepwell plate from VWR. Note the rounded wells work well with the magnet.
Load this python file to the Opentrons app: OT2_Omegabiotekfecal_v4.0.py13KB
OT2_Omegabiotekfecal_v4.0.py13KB Definition of Protocol Variables:
This protocol is written per column, best working for multiples of 8. Therefore, if you want to modify the sample number just open the script in a text editor program, and modify the following value in line 3 of the script:
"numSamps": 96 → Indicates the number of samples that you will process.
Note
This protocol is recommended for a full plate of 96 samples (12 columns). Any less columns are not optimal.
Arrange the OT-2 deck
| A | B | C | D | E | F | G | H | I | J | K | L | |
| Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | Well 7 | Well 8 | Well 9 | Well 10 | Well 11 | Well 12 | |
| Wash 3: SPM Buffer | Wash 3: SPM Buffer | Wash 3: SPM Buffer | Wash 3: SPM Buffer | EMPTY | EMPTY | EMPTY | EMPTY | EMPTY | EMPTY | EMPTY | Elution Buffer (when prompted) |
Slot 2: 96-Well PCR Plate Non-skirt, 200µl with 200uL of sample lysis on top of an empty Nest skirted PCR Plate
| A | B | C | D | E | F | G | H | I | J | K | L | |
| Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | Well 7 | Well 8 | Well 9 | Well 10 | Well 11 | Well 12 | |
| XP2 Binding Buffer and Magbind Particles Round 1 then 2 | XP2 Binding Buffer and Magbind Particles Round 1 then 2 | XP2 Binding Buffer and Magbind Particles Round 1 then 2 | EMPTY | Wash 1: VHB Buffer | Wash 1: VHB Buffer | Wash 1: VHB Buffer | Wash 1: VHB Buffer | Wash 2: SPM Buffer | Wash 2: SPM Buffer | Wash 2: SPM Buffer | Wash 2: SPM Buffer |
Note
It is possible to use Opentrons 200µL Filter Tips or Opentrons 300 Tips (as in the image below). We usually use Opentrons 200µL Filter Tips to avoid cross contamination. The tips are in fact exactly the same dimensions, except that the P200F has a filter, while the P300 does not, and is therefore able to hold more liquid.
Slot 7: Opentrons Magnetic Module with VWR 96-Well Deep Well Plate full of 300uL of sample lysis on top
Placement of LABWARE and TIPS in the OT2 Deck used for the Omega Biotek Stool extraction protocol. These materials are for purifying 96 samples.
Calibrate the deck if needed. Follow the on screen instructions.
Run the OT-2 protocol
1h 9m
Mixing Buffer and Particles with Lysed Sample Plate 1
Column 2 of tips in Slot 8 will align with column 1 in Slot 7 to mix the sample by aspirating and dispensing 10 µL . The tips will then be returned to their original starting point. Each subsequent column of tips will continue on the same pattern until all of the samples are mixed.
Allowing beads to settle on the magnet
10m
Removing the supernatant
The supernatant is removed in two steps very gently to avoid removing settled beads. Supernatant is discarded in the Liquid waste NEST 1-Well Reservoir, 195 mL in Slot 10. Column 2 of tips in Slot 8 will align with column 1 in Slot 7 to remove the supernatant and then will return the tips back to their original starting point. Each subsequent column of tips will continue on the same pattern until all of the samples have their supernatant removed.
Adding VHB Buffer to Lysed Sample Plate 1 for wash 1
Column 2 of tips in Slot 5 will transfer VHB BufferOmega BiotekCatalog #VHB-440 from wells 5-8 in Slot 4 in two 133 µL transfer steps to each sample in Lysed Sample Plate 1 in Slot 7 without touching the lysates. The tips will then be dropped into the waste container.
Mixing VHB with Lysed Sample Plate 1
Column 2 of tips in Slot 8 will align with column 1 in Slot 7 to mix the sample by aspirating and dispensing 10 µL . The tips will then be returned to their original starting point. Each subsequent column of tips will continue on the same pattern until all of the samples are mixed.
Incubating VHB Buffer and lysate
4m
Removing the supernatant from the wash
The supernatant is removed in two steps very gently to avoid removing settled beads. Supernatant is discarded in the Liquid waste NEST 1-Well Reservoir, 195 mL in Slot 10. Column 2 of tips in Slot 8 will align with column 1 in Slot 7 to remove the supernatant and then will return the tips back to their original starting point. Each subsequent column of tips will continue on the same pattern until all of the samples have their supernatant removed.
Adding SPM Buffer to Lysed Sample Plate 1 for wash 2
Column 3 of tips in Slot 5 will transfer SPM BufferOmega BiotekCatalog #SPM-300 from wells 9-12 in Slot 4 two 133 µL transfer steps to each sample in Lysed Sample Plate 1 in Slot 7 without touching the lysates. The tips will then be dropped into the waste container.
Mixing SPM with Lysed Sample Plate 1
Column 2 of tips in Slot 8 will align with column 1 in Slot 7 to mix the sample by aspirating and dispensing 10 µL . The tips will then be returned to their original starting point. Each subsequent column of tips will continue on the same pattern until all of the samples are mixed.
Incubating VHB Buffer and lysate
4m
Removing the supernatant from the wash
The supernatant is removed in two steps very gently to avoid removing settled beads. Supernatant is discarded in the Liquid waste NEST 1-Well Reservoir, 195 mL in Slot 10. Column 2 of tips in Slot 8 will align with column 1 in Slot 7 to remove the supernatant and then will return the tips back to their original starting point. Each subsequent column of tips will continue on the same pattern until all of the samples have their supernatant removed.
Adding SPM Buffer to Lysed Sample Plate 1 for wash 3
Column 4 of tips in Slot 5 will transfer SPM BufferOmega BiotekCatalog #SPM-300 from wells 1-3 in Slot 1 in two 133 µL transfer steps to each sample in Lysed Sample Plate 1 in Slot 7 without touching the lysates. The tips will then be dropped into the waste container.
Mixing SPM with Lysed Sample Plate 1
Column 2 of tips in Slot 8 will align with column 1 in Slot 7 to mix the sample by aspirating and dispensing 10 µL . The tips will then be returned to their original starting point. Each subsequent column of tips will continue on the same pattern until all of the samples are mixed.
Incubating SPM Buffer and lysate
4m
Removing the supernatant from the wash
The supernatant is removed in two steps very gently to avoid removing settled beads. Supernatant is discarded in the Liquid waste NEST 1-Well Reservoir, 195 mL in Slot 11. Column 2 of tips in Slot 8 will align with column 1 in Slot 7 to remove the supernatant and then will return the tips back to their original starting point. Each subsequent column of tips will continue on the same pattern until all of the samples have their supernatant removed.
Allowing beads to air dry
The Opentrons Magnetic Module is engaged for 00:01:00 to allow the Mag-Bind Particles CHOmega BiotekCatalog #MBPCH- 50 to air dry.
1m
Removing excess wash buffer
Column 2 of tips in Slot 8 will align with column 1 in Slot 7 to remove excess wash by aspirating 10 µL and dispensing into the Liquid waste NEST 1-Well Reservoir, 195 mL in Slot 11. The tips will then be returned to their original starting point. Each subsequent column of tips will continue on the same pattern until all of the wash buffer is removed.
Note
It is important to remove any residual wash buffer before allowing beads to dry as it contains alcohol. Alcohol could prevent a good elution in the next step and inhibit further processes.
Allowing beads to air dry
The Opentrons Magnetic Module remains engaged for 00:02:00 to allow theMag-Bind Particles CHOmega BiotekCatalog #MBPCH- 50 to have a final air dry and then disengages.
Note
Do not let beads dry for too long to prevent cracking of the pellet.
Expected result
The color of beads will change from shining dark brown to light brown when dried.
2m
Adding elution buffer to Lysed Sample Plate 1 for Elution 1
The user must remove the 2.0 mL tubes with Elution BufferOmega BiotekCatalog #PDR048 from the 70 °C incubator and pour 4 of these tubes into well 12 in Slot 1.
Column 5 of tips in Slot 5 will transfer 60 µL of Elution BufferOmega BiotekCatalog #PDR048 from well 12 in Slot 1 to column 1 in Slot 7 and mix by aspirating and dispensing 40 µL . The tips will then be dispensed into the waste container. Each subsequent column of tips will continue on the same pattern until all of the samples are mixed with warmed elution buffer.
Note
If you own a Temperature module from Opentrons you can also use it to keep the elution buffer continually warm.
Incubating the beads with DNA in elution buffer
The protocol is paused for 00:15:00 to allow for the Mag-Bind Particles CHOmega BiotekCatalog #MBPCH- 50 to incubate the DNA in the elution buffer at Room temperature .
15m
Allowing beads to settle on the magnet
The Opentrons Magnetic Module is engaged for 00:02:00 to give the Mag-Bind Particles CHOmega BiotekCatalog #MBPCH- 50 time to settle on the magnet away from the elution.
2m
Transferring each sample elution to Sample Elution Plate 1
Column 6 of tips in Slot 5 will transfer 60 µL of each eluate to a new, clean Nest skirted PCR Plate in Slot 3. Each subsequent column of tips will continue on the same pattern until all of the sample eluates are transferred, extending into the tips in Slot 9. The tips will be returned to the tip boxes to be reused for Elution 2.
Adding elution buffer to Lysed Sample Plate 1 for Elution 2
The user must remove the 2.0 mL tubes with Elution BufferOmega BiotekCatalog #PDR048 from the 70 °C incubator and pour the remaining 2 tubes into well 12 in Slot 1. They must also replace the 96-Well PCR Plate Non-skirt, 200µl now empty of sample lysis on top of an empty Nest skirted PCR Plate in Slot 2 with a new, clean Nest skirted PCR Plate to receive Elution 2
Column 5 of tips in Slot 9 will transfer 40 µL of Elution BufferOmega BiotekCatalog #PDR048 from well 12 in Slot 1 to column 1 in Slot 7 and mix by aspirating and dispensing 20 µL . The tips will then be dispensed into the waste container. Each subsequent column of tips will continue on the same pattern until all of the samples are mixed with warmed buffer, extending into the tips in Slot 6.
Incubating the beads with DNA in elution buffer
The protocol is paused for 00:15:00 to allow for theMag-Bind Particles CHOmega BiotekCatalog #MBPCH- 50 to incubate the DNA in the buffer at Room temperature .
15m
Allowing beads to settle on the magnet
The Opentrons Magnetic Module is engaged for 00:02:00 to give the Mag-Bind Particles CHOmega BiotekCatalog #MBPCH- 50 time to settle on the magnet away from the elution.
2m
Transferring each sample elution to Sample Elution Plate 2
The same tips that were used for Elution 1 will transfer 40 µL of each eluate to a new, clean Nest skirted PCR Plate in Slot 2. Each subsequent column of tips will continue on the same pattern until all of the sample eluates are transferred. The tips will be dispensed into the waste container.
Storage of Sample Elution Plates 1 and 2
Cover the plates with an aluminium plate seal and store at 4 °C for use or -20 °C for long term storage.
QC of Sample Elution Plates 1 and 2
See QC Note in Step 1 for options.
After finishing the protocol
1h 9m
Clean the OT2 deck and walls with:
10% Bleach 1 rinse
Distilled Water 1 rinse
70% Alcohol 2 rinses
Note
Avoid wetting any electronic parts.
Clean OT2 module with:
70% Alcohol 2 rinses
Note
Avoid wetting electronic parts.
Air dry OT2 robot and modules.
Protocol references