TITLE: ISL Opentrons  pipeline:  gDNA bead cleanup
AUTHORS: Jhakelin Reyes Vasquez, P. Sánchez-Vendizú, Kristina N Vsevolodova, Gideon Erkenswick, Mrinalini Watsa

[DESCRIPTION]
This protocol is an automated pipeline to clean a plate of extracted DNA using SPRI bead cleanup. It is functional for both. It is typically used to clean up a portion of extracted DNA which has suspected impurities capable of affecting downstream PCR or sequencing protocols. This is often a side effect of extraction by MagBeads with no spin-column based purifications. We have found this protocol to be effective on DNA extracted from blood, biopsy and feces in a wide range of Neotropical vertebrates. 

This protocol was developed and optimised for the following:
Platform: Opentrons OT-2 automated pipetting robot
Kit: Ampure beads and home-brewed bead solutions
Tips Used: 5 boxes (2 x 200uL Opentrons Filtered Tip boxes and 3 x 20uL Opentrons Filtered Tip boxes)
Number of samples: 96 

V2 - This is a new version of the original protocol modified as follows:
We include an updated version of the protocol python file, which is optimized to work on version 6.3.1. Note Version 1 protocol's file is likely to glitch, so please use this file instead.
To assist with implementing this new protocol in your lab, we also include a simplified version of the python file for a test water run in Section 2. The test reduces incubation times, and works for samples in multiples of 8, so you can test only 8 for a quicker version of the protocol.
Error correction: OT-2 Single Channel Electronic Pipette P20 was changed to OT-2 8-Channel Electronic Pipette P20.
Starting sample clean up volume was changed to 25uL from 15uL and all subsequent calculations and descriptions were changed to reflect this. Note: You can alter these volumes easily as well.
Bead ratio was changed to 1.5X and all subsequent calculations and descriptions were changed to reflect this.Note: this can also be altered to match your specific use case.



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

 


[GUIDELINES]
There are few things to consider with this protocols:

Step 1.1: In our experience, DNA recovery following SPRI bead cleanups is higher when total volumes used for the incubation steps are higher. This, however, does cost more in terms of beads. If you can afford it in your protocol though, always augment your DNA needing cleanup to 100-200uL, and add beads in a 1.2 ratio to those volumes. Final elution can remain in a small volume.

Step 2: 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 6.3.1


[STEPS]
SECTION: BEFORE STARTING
1. Materials:
Autoclave and UV the items you will use to ensure sterility. Some items can be autoclaved and reused as indicated below.
 
      ItemquantitycheckstatusAutoclavedUVOpentrons 20µL Filter Tips31NEWOpentrons 200µL Filter Tips21NEW1NEST 1-Well Reservoirs, 195 mL11REUSED11NEST 12-Well Reservoirs, 15 mL11REUSED11Nest skirted PCR Plate11NEW1Denville non-skirted PCR Plate (200uL)11NEW01 plastic seal11NEW012ml microcentrifugue tubes 21NEW11

Opentrons Equipment List
 
On the right pipette mount use the P300M
 
On the left pipette mount use the P20M
 


SECTION: BEFORE STARTING
2. Before loading your protocol, load this labware file into your Opentrons app:   

This labware definition allows us to use a nonskirted plate in the Opentrons 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 ). 

Load this python file to the Opentrons app:   

To test the protocol before starting, there is a modified version of the python file with shortened incubation times and minimized sample total to input into the app if you wish to do so:   

SECTION: BEFORE STARTING
1.1. Reagents:

Prepare all reagents in advance:
 
  
3.   , freshly prepared
4.    


 



We begin with a cleanup sample volume of 25uL. This allows you to cleanup a small volume, but larger volumes are easily cleaned with the same protocol. Simply add water 1:1, and then proceed.


 

 


SECTION: BEFORE STARTING
1.2. Add water in a 1:1 ratio by volume of water to samples. 

 


SECTION: BEFORE STARTING
1.3. Add beads in a 1:1.5 ratio (sample: beads) allowing for the fact that the sample contains both sample +water from step 2.1.

 

SECTION: OT2 SCRIPT DEFINITIONS
3. Definition of samples and labwares:
SECTION: BEFORE STARTING
1.4. Each sample will be washed twice with freshly prepared Ethanol 70%. 

 

SECTION: OT2 SCRIPT DEFINITIONS
3.1. gTNA samples
Samples that will be cleaned and are in the elution plate A from previous TNA extraction.

Position: Slot 2, Nest skirted PCR Plate with TNA 
Name in the Deck: gTNA plate elution A
Labware name in the protocol: gTNA_plate_A
Sample name in the script: gTNA_samples

SECTION: OT2 Clean up Protocol
5. Protocol

SECTION: OT2 SCRIPT DEFINITIONS
3.2. Samples to be cleaned
Samples that are in the magnet to be cleaned.

Position: Slot 1, Nest skirted PCR Plate in Opentrons Magnetic Module
Name in the Deck: to_be_cleaned gTNA plate
Labware name in the script protocol: mag_plate
Sample name in the script: samples_to_be_cleaned

SECTION: OT2 SCRIPT DEFINITIONS
3.3. Cleaned samples
Samples that have been cleaned and will be eluted in the clean up plate.

Position: Slot 6, Axygen™ PCR Tube Storage Rack with 96-Well PCR Plate Non-skirt, 200µl 
Name in the Deck: cleaned_up gDNA plate
Labware name in the protocol: clean_up_plate
Sample name in the script: cleaned_samples

SECTION: After finishing script
None. Clean OT2 module with:
  2 rinses

 

SECTION: After finishing script
None. Dry OT2 robot and modules very well.
SECTION: After finishing the protocol
6. Clean the OT2 deck and walls with: 
  1 rinse 
   1 rinse 
  2 rinses
 


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

 

SECTION: After finishing the protocol
8. Air dry OT2 robot and modules.
SECTION: OT2 SCRIPT DEFINITIONS
4. Protocol variables definition

This protocol is written to use 25 µL  of gTNA sample + 25 µL  of water to reach 50 µL  as final volume to start the clean up process for 96 samples. Therefore, if you want to modify the volumes and sample number just open the script in a text editor program, search and modify the values in the third line of the script:

"sample_number":  96  → Indicates the number of samples that you will process.

 

"gTNA_volume":     15   → Volume of gDNA that will be cleaned.
"bead_ratio":          1.5  → Ratio of beads volume 
"elution_buffer_volume": 15  → Volume of water for elution
 

"incubation_time":  6 → Time in minutes for incubation of beads with the sample
"pelleting_time":       6 → Time with magnet engaged.
"drying_time":         5 → Time to the let alcohol evaporate

SECTION: OT2 Clean up Protocol
5.1. Transferring water to the gTNA plate

25 µL of   is transferred from Well 9 in the NEST 12-Well Reservoirs, 15 mL in Slot 3 to each column of a new Nest skirted PCR Plate placed in the Opentrons Magnetic Module in Slot 1. 
The first column of tips in Slot 11 is used for dispensing water to all the columns.
SECTION: OT2 Clean up Protocol
5.2. Transferring gTNA to the  gTNA plate

25 µL of   is transferred from the gTNA plate elution A in Slot 2 to a the to_be_cleaned gTNA plate in  Slot 1. 
Tips in  Slot 11 are used for this step. 
 

SECTION: OT2 Clean up Protocol
5.3. Dispensing SPRI  beads

75 µL of SPRI  beads are dispensed from Well 1 in the NEST 12-Well Reservoirs, 15 mL in Slot 3 to the to_be_cleaned gTNA plate in Slot 1. 
The first column of tips in Slot 5 is used for dispensing SPRI beads to all the columns.
SECTION: OT2 Clean up Protocol
5.4. Mixing samples and beads

Two mixing steps are defined in the script. The first column will be mixed, then the second and so on to the 12th column, then it will be repeated. The whole process is approximately 15 min long.
Tips in Slot 4 are used for this step. Each column of tips is used to mix each column of samples.
 

SECTION: OT2 Clean up Protocol
5.5. Allowing beads to settle on the magnet 

The Opentrons Magnetic Module is engaged for 6 min to allow beads settle.
SECTION: OT2 Clean up Protocol
5.6. 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.
Tips in Slot 4 are used for this step. Each column of tips is used for one column of samples.
SECTION: OT2 Clean up Protocol
5.7. The first washing step

100 µL of   is dispensed for washing beads from Well 5 in the NEST 12-Well Reservoirs, 15 mL in Slot 3 to the to_be_cleaned gTNA plate in Slot 1.
The second column of tips in Slot 5 is used for dispensing SPRI beads to all the columns.
SECTION: OT2 Clean up Protocol
5.8. Removing the 1st ethanol wash

After an incubation of 30 s, the supernatant is removed in two steps very gently to avoid removing settled beads. Supernatant is discarded in the liquid waste NEST 1-Well Reservoirs, 195 mL in Slot 10.
Tips in Slot 4 are used for this step. Each column of tips is used for one column of samples.
SECTION: OT2 Clean up Protocol
5.9. The second washing step

100 µL of   is dispensed for washing beads from Well 6 in the NEST 12-Well Reservoirs, 15 mL in Slot 3 to the to_be_cleaned gTNA plate in the Slot 1.
The second column of tips in Slot 5 is used for dispensing SPRI beads to all the columns.
SECTION: OT2 Clean up Protocol
5.10. Removing the 2nd ethanol wash

After an incubation of 30 s, the supernatant is removed in two steps very gently to avoid removing settled beads. Supernatant is discarded in the Liquid waste NEST 1-Well Reservoirs, 195 mL in Slot 10.
Tips in Slot 4 are used for this step. Each column of tips is used for one column of samples.
SECTION: OT2 Clean up Protocol
5.11. Removing any remaining ethanol

30 µL of remaining ethanol is removed very gently to avoid removing settled beads. Supernatant is discarded in Liquid waste NEST 1-Well Reservoirs, 195 mL in Slot 10.
Tips in Slot 4 are used for this step. Each column of tips is used for one column of samples.
 

SECTION: OT2 Clean up Protocol
5.12. Drying the beads

A pause of 5 min is set to allow beads to dry at Room temperature to evaporate remaining ethanol.
 

 


SECTION: OT2 Clean up Protocol
5.13. Adding elution buffer or water

Disengaged the  Opentrons Magnetic Module.
19 µL of   are transferred from the Well 9 in the NEST 12-Well Reservoirs, 15 mL in the Slot 3 to each column of the cleaning_up gTNA plate in the Slot 1. Samples are mixed after adding water.
Tips in the Slot 7 are used for this step. Each column of tips is used for each column of samples. 

SECTION: OT2 Clean up Protocol
5.14. Mixing the beads

This is the second mixing step of water and sample before elution. The first column will be mixed, then the second and so on to the 12th column
The whole process is approximately 10 min long.
Tips in the Slot 7 are used for this step.
SECTION: OT2 Clean up Protocol
5.15. Binding beads to the magnet

The Opentrons Magnetic Module is engaged for 6 min to allow beads to pellet.
SECTION: OT2 Clean up Protocol
5.16. Elution of final DNA

19 µL of cleaned gTNA are transferred from the to_be_cleaned gTNA plate in  Slot 1 to the 
 cleaned_up gTNA plate in Slot 6.
Tips in the Slot 8 are used for this step. Each column of tips is used for each column of samples. 
SECTION: OT2 Clean up Protocol
5.17. Storage of cleaned gDNA

Cover the cleaned_up gTNA plate with a plate seal and store at 4 °C  for use or -20 °C  for long term storage.

SECTION: BEFORE STARTING
2.1. Arrange the OT-2 deck 

Number of samples: 96


Slot 1: Opentrons Magnetic Module with Nest skirted PCR Plate empty (to receive cleaned DNA)
Slot 2: Nest skirted PCR Plate with TNA 
Slot 3: NEST 12-Well Reservoirs, 15 mL with reagents preloaded in the following order:
 
            123456789101112SPRI BEADSALCOHOLWATER
 
Slot 4: Opentrons 200µL Filter Tips

 

Slot 5: Opentrons 200µL Filter Tips
Slot 6:  Axygen™ PCR Tube Storage Rack with 96-Well PCR Plate Non-skirt, 200µl 


 

Slot 8: Opentrons 200µL Filter Tips
Slot 9: Opentrons 200µL Filter Tips
Slot 10: NEST 1-Well Reservoirs, 195 mL (for waste)
Slot 11: Opentrons 200µL Filter Tips

 
 
SECTION: BEFORE STARTING
2.2. Calibrate the deck. Follow the onscreen instructions.