Opentrons pipeline: gDNA bead cleanup

Jhakelin Reyes Vasquez, P. Sánchez-Vendizú, Gideon Erkenswick, Mrinalini Watsa

Published: 2023-09-26 DOI: 10.17504/protocols.io.3byl4j9pzlo5/v1

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

Before start

Clean the OT2 deck and walls with:

1 rinse

2 rinses

Note

Avoid wetting the electronic parts.

Steps

BEFORE STARTING

Materials:

Autoclave and UV the items you will use to ensure sterility. Some items can be autoclaved and reused as indicated below.

A	B	C
Item	#	Status
Opentrons 200µL Filter Tips	2	NEW
Opentrons 20µL Filter Tips	3	NEW
NEST 1-Well Reservoirs, 195 mL	1	REUSED
NEST 12-Well Reservoirs, 15 mL	1	REUSED
Nest skirted PCR Plate	1	NEW
96-Well PCR Plate Non-skirt, 200µl	1	NEW
Axygen™ PCR Tube Storage Racks	1	NEW
Polyester plate seal	1	NEW
2ml microcentrifugue tubes	2	NEW

Opentrons Equipment List

Equipment

Value	Label
OT-2	NAME
Liquid handler	TYPE
Opentrons	BRAND
OT-2	SKU
https://opentrons.com/	LINK

On the right pipette mount use the P300M

Equipment

Value	Label
OT-2 8 Channel Electronic Pipette	NAME
Pipette	TYPE
Opentrons	BRAND
P300M	SKU

On the left pipette mount use the P20M

Equipment

Value	Label
OT-2 Single Channel Electronic Pipette	NAME
Pipette	TYPE
Opentrons	BRAND
P20S	SKU

1.1.

Reagents:

Prepare all reagents in advance:

, freshly prepared

Note

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

We begin with a cleanup sample volume of 15uL. 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.

A	B	C	D
Ingredient	Amount per sample	Amount per 96 samples	Notes
Water	15	=15*96
SPRI beads	=1521.2	=1521.2*96	1.2x water+sample
70% ethanol	250	=250*96

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

1.2.

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 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 in the position detailed in the Step 3 just before dispensing. It should be warmed to 55°C

1.3.

Add beads in a 1: 1.2 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

1.4.

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: denville_96_axygenbase_200ul.json

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: cleanup_gDNA_v4.3.py

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:

A	B	C	D	E	F	G	H	I	J	K	L	M
Channel #	1	2	3	4	5	6	7	8	9	10	11	12
Contents	SPRI BEADS				ALCOHOL	ALCOHOL			WATER

Ingredients plan for a 12-well reservoir

Slot 4: Opentrons 200µL Filter Tips

Note

It is possible to use 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. or Opentrons 300 Tips (as in the image below). We usually use 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. 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.

2.2.

Calibrate the deck. Follow the onscreen instructions.

OT2 SCRIPT DEFINITIONS

Definition of samples and labwares:

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

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

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

Protocol variables definition

This protocol is written to use 15µL of gTNA sample + 15µL of water to reach 30µL 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": 1.2 → 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

Protocol

5.1.

Transferring water to the gTNA plate

15µL 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.

5.10.

Removing the 2nd ethanol wash

After an incubation of 0h 0m 30s, 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.

5.11.

Removing any remaining ethanol

30µL 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.

5.12.

Drying the beads

A pause of 0h 5m 0s is set to allow beads to dry at Room temperature to evaporate remaining ethanol.

Note

Do not let beads dry for too long to prevent cracking of the pellet

Citation

The color of beads will change from shining dark brown to light brown when dried.

5.13.

Adding elution buffer or water

Disengaged the Opentrons Magnetic Module.

15µL 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.

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 0h 10m 0s long.

Tips in the Slot 7 are used for this step.

5.15.

Binding beads to the magnet

The Opentrons Magnetic Module is engaged for 0h 6m 0s to allow beads to pellet.

5.16.

Elution of final DNA

15µL 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.

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.

5.2.

Transferring gTNA to the gTNA plate

15µL 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.

Note

Samples are mixed in this step before transferring, in a programmed mixing step.

5.3.

Dispensing SPRI beads

36µL 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.

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 0h 15m 0s 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.

5.5.

Allowing beads to settle on the magnet

The Opentrons Magnetic Module is engaged for 0h 6m 0s to allow beads settle.

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.

5.7.

The first washing step

100µL 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.

5.8.

Removing the 1st ethanol wash

After an incubation of 0h 0m 30s, 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.

5.9.

The second washing step

100µL of are 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.

After finishing the protocol

Clean the OT2 deck and walls with:

1 rinse

2 rinses

Note

Avoid wetting any electronic parts.

Clean OT2 module with:

2 rinses

Note

Avoid wetting electronic parts.

Air dry OT2 robot and modules.

Opentrons pipeline: gDNA bead cleanup

Abstract

Before start

Steps

BEFORE STARTING

OT2 SCRIPT DEFINITIONS

OT2 Clean up Protocol

After finishing the protocol

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