Oct 21, 2023

Public workspaceISL Opentrons pipeline: gDNA bead cleanup V.2

DOI
dx.doi.org/10.17504/protocols.io.3byl4j9pzlo5/v2
  • 1Imperial College London;
  • 2Conservación Amazónica-ACCA;
  • 3Universidad Austral de Chile;
  • 4Universidad Nacional Mayor de San Marcos;
  • 5Sn Diego Zoo Wildlif Alliance;
  • 6Field Projects International;
  • 7San Diego Zoo Wildlife Alliance
lalabmanager
Open access
DOI: dx.doi.org/10.17504/protocols.io.3byl4j9pzlo5/v2
Protocol Citation: Jhakelin Reyes Vasquez, P. Sánchez-Vendizú, Kristina N Vsevolodova, Gideon Erkenswick, Mrinalini Watsa 2023. ISL Opentrons pipeline: gDNA bead cleanup. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4j9pzlo5/v2Version created by Mrinalini Watsa
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
We use this protocol and it's working
Created: October 17, 2023
Last Modified: October 21, 2023
Protocol Integer ID: 89475
Keywords: gDNA clean up, DNA SPRI beads, Automated Opentrons pipeline, bead cleanup, opentrons, insitulabs
Funders Acknowledgement:
Gordon and Betty Moore Foundation
Grant ID: 9776
Gordon and Betty Moore Foundation
Grant ID: 9772
Abstract
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.


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

Materials
Protocol materials
ReagentNuclease-free Water
Step 1.1
Reagent70% Alcohol
In Before starting and 3 steps
Reagent10% Bleach
Before starting, Step 6
ReagentAgencourt AmPure XP beadsCatalog #A63880
Step 1.1
ReagentgTNA
Step 5.2
ReagentSolutions for Purifying DNA by solid-phase reversible immobilization (SPRI) lab-made
Step 1.1
ReagentDistilled Water
Before starting, Step 6
ReagentEthanol 70% [Note: freshly prepared]
In 2 steps
ReagentUltrapure Distilled, Nuclease Free Water
In 2 steps
Safety warnings
Attention
Only the standard warnings apply - use PPE to ensure sterility. No ingredients used here are hazardous.
Before start
Clean the OT2 deck and walls with:
Reagent10% Bleach 1 rinse
ReagentDistilled Water 1 rinse
Reagent70% Alcohol 2 rinses

Note
Avoid wetting the electronic parts.

BEFORE STARTING
BEFORE STARTING
Materials:
Autoclave and UV the items you will use to ensure sterility. Some items can be autoclaved and reused as indicated below.
ABC
Item#Status
Opentrons 200µL Filter Tips2NEW
Opentrons 20µL Filter Tips3NEW
NEST 1-Well Reservoirs, 195 mL1REUSED
NEST 12-Well Reservoirs, 15 mL1REUSED
Nest skirted PCR Plate1NEW
96-Well PCR Plate Non-skirt, 200µl1NEW
Axygen™ PCR Tube Storage Racks1NEW
Polyester plate seal1NEW
2ml microcentrifugue tubes 2NEW

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
https://shop.opentrons.com/8-channel-electronic-pipette/
LINK
On the left pipette mount use the P20M
Equipment
OT-2 8-Channel Electronic Pipette P20
NAME
Pipette
TYPE
Opentrons
BRAND
P20M
SKU
https://shop.opentrons.com/8-channel-electronic-pipette/
LINK


Reagents:

Prepare all reagents in advance:
  1. ReagentNuclease-free WaterContributed by users
  2. ReagentSolutions for Purifying DNA by solid-phase reversible immobilization (SPRI) lab-madeContributed by users
3. Reagent70% AlcoholContributed by users , freshly prepared
4. ReagentAgencourt AmPure XP beadsContributed by usersCatalog #A63880


Note
You can also make your own bead solution such as from this protocol on protocols.io



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.


ABCD
IngredientAmount per sampleAmount per 96 samplesNotes
Water252400
SPRI beads3672001.5x water+sample
70% ethanol25024000

Note
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 1.5 ratio to those volumes. Final elution can remain in a small volume.


Add water in a 1:1 ratio by volume of water to samples.

Note
This reagent will be put in the NEST 12-Well Reservoirs, 15 mL in the position detailed in the Step 3 just before dispensing. It should be warmed to Temperature55 °C


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.

Note
This reagent will be put in the NEST 12-Well Reservoirs, 15 mL in the position detailed in Step 3

Each sample will be washed twice with freshly prepared Ethanol 70%.

Note
This reagent will be put in the NEST 12-Well Reservoirs, 15 mL in the position detailed in the Step 3

Before loading your protocol, load this labware file into your Opentrons app: Download denville_96_axygenbase_200ul.jsondenville_96_axygenbase_200ul.json25KB

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: Download cleanup_gDNA_v4.4.pycleanup_gDNA_v4.4.py12KB

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: Download cleanup_gDNA_v4.4.w.pycleanup_gDNA_v4.4.w.py12KB

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:
ABCDEFGHIJKLM
Channel #123456789101112
ContentsSPRI BEADSALCOHOLALCOHOLWATER
Ingredients plan for a 12-well reservoir
Slot 4: Opentrons 200µL Filter Tips

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


Note
Here, we use more affordable nonskirted plates over the NEST plates because they are a) cheaper and b) 200uL and able to hold more volume. By placing the plate inside the storage rack, we created a way for a nonskirted plate to be "clipped" into the deck. Together, they have a new labware definition titled: MISSING ADD LABWARE DEFINITION.

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

Placement of LABWARE and TIPS in the OT2 Deck used for the gDNA clean up protocol. These materials are for cleaning 96 samples.
Calibrate the deck. Follow the onscreen instructions.
OT2 SCRIPT DEFINITIONS
OT2 SCRIPT DEFINITIONS
Definition of samples and labwares:
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

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

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

Protocol variables definition

This protocol is written to use Amount25 µL of gTNA sample + Amount25 µL of water to reach Amount50 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.

Note
It is better if it is a multiple of 8

"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
Note
This is the same volume as gTNA cleaned.
You can also set it to be 1ul more than the desired volume to avoid losing beads

"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

OT2 Clean up Protocol
OT2 Clean up Protocol
2h 11m
Protocol

Transferring water to the gTNA plate

Amount25 µL of ReagentUltrapure Distilled, Nuclease Free Water 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.
8m
Transferring gTNA to the gTNA plate

Amount25 µL of ReagentgTNA 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.
Note
Samples are mixed in this step before transferring, in a programmed mixing step.

8m
Dispensing SPRI beads

Amount75 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.
8m
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 Duration00:15:00 m long.
Tips in Slot 4 are used for this step. Each column of tips is used to mix each column of samples.
Note
Make sure samples are well mixed, samples should have an homogeneous color.

15m
Allowing beads to settle on the magnet

The Opentrons Magnetic Module is engaged for Duration00:06:00 m to allow beads settle.
6m
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.
10m
The first washing step

Amount100 µL of ReagentEthanol 70% [Note: freshly prepared] 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.
8m
Removing the 1st ethanol wash

After an incubation of Duration00:00:30 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.
10m
The second washing step

Amount100 µL of ReagentEthanol 70% [Note: freshly prepared] 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.
8m
Removing the 2nd ethanol wash

After an incubation of Duration00:00:30 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.
10m
Removing any remaining ethanol

Amount30 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.
Note
It is important to remove any residual ethanol before allowing beads to dry. Alcohol could prevent a good elution in the next step and inhibit further processes.

8m
Drying the beads

A pause of Duration00:05:00 m is set to allow beads to dry at TemperatureRoom temperature to evaporate remaining ethanol.
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.



5m
Adding elution buffer or water

Disengaged the Opentrons Magnetic Module.
Amount19 µL of ReagentUltrapure Distilled, Nuclease Free Water 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.

10m
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 Duration00:10:00 m long.
Tips in the Slot 7 are used for this step.
10m
Binding beads to the magnet

The Opentrons Magnetic Module is engaged for Duration00:06:00 m to allow beads to pellet.
6m
Elution of final DNA

Amount19 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.
8m
Storage of cleaned gDNA

Cover the cleaned_up gTNA plate with a plate seal and store at Temperature4 °C for use or Temperature-20 °C for long term storage.

After finishing the protocol
After finishing the protocol
Clean the OT2 deck and walls with:
Reagent10% BleachContributed by users 1 rinse
ReagentDistilled WaterContributed by users 1 rinse
Reagent70% AlcoholContributed by users 2 rinses
Note
Avoid wetting any electronic parts.


Clean OT2 module with:
Reagent70% AlcoholContributed by users 2 rinses

Note
Avoid wetting electronic parts.

Air dry OT2 robot and modules.
Protocol references
If you want to make your own beads, we recommend this protocol: dx.doi.org/10.17504/protocols.io.bnz4mf8w