TITLE: Opentrons Pipeline: DNA Extraction with the Omega Biotek Stool Kit 
AUTHORS: Kristina Vsevolodova, Gideon Erkenswick, Mrinalini Watsa

[DESCRIPTION]
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 


[BEFORE_START]
Clean the OT2 deck and walls with: 
  1 rinse 
   1 rinse 
  2 rinses

 


[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.


[STEPS]
SECTION: Before starting
2. Materials

5x Nest 50mL Falcon Tubes for holding accurate reagent amounts before starting protocol.
4x Opentrons 200µL Filter Tips
1x VWR 96 Deep Well Plates 1mL for 300µL of lysate.
2x NEST 1-Well Reservoirs, 195 mL for extraction waste collection during protocol.
2x NEST 12-Well Reservoirs, 15 mL for holding reagents during protocol.
1x 96-Well PCR Plate Non-skirt, 200µl for 200µL of lysate.
3x Nest skirted PCR Plate for holding non-skirt plate and for final elutions.
2x Aluminium Seals
6x 2mL Tubes
100-1000µL pipette
1000µL pipette tips
Incubator or water bath that can reach 70°C or more
6.1. 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.

 

SECTION: OT-2 script definitions
5. Definition of samples and labware:
SECTION: OT-2 script definitions
5.1. 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
SECTION: Prepare the OT-2
6. Before loading your protocol, load the following labware files into your Opentrons app:   
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). 


  
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:   

SECTION: OT-2 script definitions
5.2. 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
SECTION: OT-2 script definitions
5.3. Sample Elution 1
Samples that have been eluted from the beads for the first round elution 1. 

Position: Slot 3, Empty Nest skirted PCR Plate (to receive 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
SECTION: OT-2 script definitions
5.4. 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
SECTION: Before starting
1. Ingredients

 
 
  (Contains everything for extraction including lysis reagents)

OR separate reagents (not including lysis reagents): 

 
  
  
  
  

 

SECTION: Before starting
1.1. For the 1x96 Kit

Dilute SPM Buffer with 70 mL   per bottle and store at Room temperature.

Dilute VHB Buffer with 28 mL    per bottle and store at Room temperature.

SECTION: Before starting
2.1. 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. 
SECTION: Before starting
1.2. For the 4x96 Kit

Dilute SPM Buffer with 70 mL    per bottle and store at Room temperature.

Dilute VHB Buffer with 112 mL   per bottle and store at Room temperature.

SECTION: Prepare reagents
4. After the reagents are properly diluted and materials are ready, prepare the following amounts:
 
   Item NameAmount per sample [uL]96 samplesMagbind Bead Particles CH201920XP2 Binding Buffer60057600VHB Buffer40038400SPM Buffer80076800Elution Buffer1009600

 
SECTION: Prepare reagents
4.1. Fill one Nest 50mL Falcon Tube with the amount of   Round 1 and   Round 1 required in the table, and mix well. Keep at Room temperature. Split between two tubes if needed.

SECTION: Prepare reagents
4.3. Fill one Nest 50mL Falcon Tube with the volume indicated in the table with wash 1:  . Keep at Room temperature.

SECTION: Prepare reagents
4.2. Fill one Nest 50mL Falcon Tube with the amount of   Round 2 and   Round 2 required in the table, and mix well. Keep at Room temperature. Split between two tubes if needed.
SECTION: Prepare reagents
4.4. Fill one Nest 50mL Falcon Tube with the volume indicated in the table with wash 2:  . Keep at Room temperature.

SECTION: Prepare reagents
4.5. Fill one Nest 50mL Falcon Tube with the volume indicated in the table with wash 3:  . Keep at Room temperature.
SECTION: Prepare reagents
4.6. Distribute   between each 2mL tube evenly.
SECTION: Prepare reagents
4.7. Set an incubator or water bath to 70 °C and heat  in the incubator to 70 °C.

7. Arrange the OT-2 deck
7.1. Slot 1:  NEST 12-Well Reservoirs, 15 mL with reagents preloaded in the following order:
            DECK 4: NEST 12 well containerWell 1Well 2Well 3Well 4Well 5Well 6Well 7Well 8Well 9Well 10Well 11Well 12XP2 BINDING BUFFER + BEADSFREE SPACEVHB BUFFERSPM BUFFER
Slot 2: 96-Well PCR Plate Non-skirt, 200µl with 200uL of sample lysis on top of an empty Nest skirted PCR Plate
Slot 3: Empty Nest skirted PCR Plate (to receive elution 1)
Slot 4: NEST 12-Well Reservoirs, 15 mL with reagents preloaded in the following order:
            DECK 4: NEST 12 well containerWell 1Well 2Well 3Well 4Well 5Well 6Well 7Well 8Well 9Well 10Well 11Well 12XP2 BINDING BUFFER + BEADSFREE SPACEVHB BUFFERSPM BUFFER
Slot 5:  Opentrons 200µL Filter Tips

 

Slot 6: Opentrons 200µL Filter Tips
Slot 7: Opentrons Magnetic Module with VWR 96-Well Deep Well Plate full of 300uL of sample lysis on top
Slot 8: Opentrons 200µL Filter Tips
Slot 9: Opentrons 200µL Filter Tips
Slot 10: NEST 1-Well Reservoirs, 195 mL (for waste)
Slot 11: NEST 1-Well Reservoirs, 195 mL (for waste)
 
7.2. Calibrate the deck if needed. Follow the on screen instructions.
SECTION: OT-2 Protocol
None. Adding Lysed Sample Plate 2 lysate to Lysed Sample Plate 1

Column 2 of tips in Slot 8 will be used transfer 200 µL of the lysate in Slot 2 to Lysed Sample Plate 1 in Slot 7 and mis 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 lysates have been transferred.

SECTION: Run the OT-2 protocol
7.3. 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. 
SECTION: Run the OT-2 protocol
7.4. Allowing beads to settle on the magnet

The Opentrons Magnetic Module is engaged and incubates the mixed samples for 10 min.
SECTION: Run the OT-2 protocol
7.5. 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.
SECTION: Run the OT-2 protocol
7.6. Adding VHB Buffer to Lysed Sample Plate 1 for wash 1

Column 2 of tips in Slot 5 will transfer   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.

SECTION: Run the OT-2 protocol
7.7. 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. 
SECTION: Run the OT-2 protocol
7.8. Incubating VHB Buffer and lysate

The Opentrons Magnetic Module is engaged and incubates the mixed samples for 4 min.
SECTION: Run the OT-2 protocol
7.9. 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.
SECTION: Run the OT-2 protocol
7.10. Adding SPM Buffer to Lysed Sample Plate 1 for wash 2

Column 3 of tips in Slot 5 will transfer   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.

SECTION: Run the OT-2 protocol
7.11. 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. 
SECTION: Run the OT-2 protocol
7.12. Incubating VHB Buffer and lysate

The Opentrons Magnetic Module is engaged and incubates the mixed samples for 4 min.
SECTION: Run the OT-2 protocol
7.13. 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.
SECTION: Run the OT-2 protocol
7.14. Adding SPM Buffer to Lysed Sample Plate 1 for wash 3

Column 4 of tips in Slot 5 will transfer   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.

SECTION: Run the OT-2 protocol
7.15. 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. 
SECTION: Run the OT-2 protocol
7.16. Incubating SPM Buffer and lysate

The Opentrons Magnetic Module is engaged and incubates the mixed samples for 4 min.
SECTION: Run the OT-2 protocol
7.17. 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.
SECTION: Run the OT-2 protocol
7.18. Allowing beads to air dry

The Opentrons Magnetic Module is engaged for 1 min to allow the   to air dry.

SECTION: Run the OT-2 protocol
7.19. 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.

 

SECTION: Run the OT-2 protocol
7.20. Allowing beads to air dry

The Opentrons Magnetic Module remains engaged for 2 min to allow the  to have a final air dry and then disengages.

 

 


SECTION: Run the OT-2 protocol
7.21. Adding elution buffer to Lysed Sample Plate 1 for Elution 1

The user must remove the 2.0 mL tubes with   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   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.
 


SECTION: Run the OT-2 protocol
7.22. Incubating the beads with DNA in elution buffer

The protocol is paused for 15 min to allow for the   to incubate the DNA in the elution buffer at Room temperature.

SECTION: Run the OT-2 protocol
7.24. 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.

The Opentrons Magnetic Module is disengaged.
SECTION: Run the OT-2 protocol
7.25. Adding elution buffer to Lysed Sample Plate 1 for Elution 2

The user must remove the 2.0 mL tubes with   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   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.

SECTION: Run the OT-2 protocol
7.26. Incubating the beads with DNA in elution buffer

The protocol is paused for 15 min to allow for the  to incubate the DNA in the buffer at Room temperature.
SECTION: Run the OT-2 protocol
7.27. Allowing beads to settle on the magnet

The Opentrons Magnetic Module is engaged for 2 min to give the   time to settle on the magnet away from the elution.

SECTION: Run the OT-2 protocol
7.28. 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.

The Opentrons Magnetic Module is disengaged.
SECTION: Run the OT-2 protocol
7.29. 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.

SECTION: Run the OT-2 protocol
7.23. Allowing beads to settle on the magnet

The Opentrons Magnetic Module is engaged for 2 min to give the   time to settle on the magnet away from the elution.
SECTION: Run the OT-2 protocol
7.30. QC of Sample Elution Plates 1 and 2

See QC Note in Step 1 for options.

SECTION: After finishing the protocol
8. Clean the OT2 deck and walls with: 
  1 rinse 
   1 rinse 
  2 rinses
 


SECTION: After finishing the protocol
9. Clean OT2 module with:
  2 rinses

 

SECTION: After finishing the protocol
10. Air dry OT2 robot and modules.
3. Opentrons Equipment List
 
On the right pipette mount use the P300M
 
Magnetic Module to place in Slot 7