Opentrons Pipeline: DNA Extraction with the Mag-Bind Blood & Tissue DNA HDQ 96 Kit Tissue Protocol
Thalía Silvestre
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment.
Abstract
This protocol is an automated pipeline to extract a full 96-well plate of DNA from tissue lysates.
This protocol was developed and optimized for the following:
Platform: Opentrons OT-2 automated pipetting robot
Kit: Mag-B®nd‱ Blood & Tissue DNA HDQ Prefilled 96 Kit
Recommended number of samples: 96
Two ellutions: Firts ellution( 60μL ) , Second ellution ( 30μL)
Steps
Samples Lysis
Preparate 96 samples lysis following the Mag-Bind® Blood & Tissue DNA HDQ 96 Kit Tissue Protocol:
Mince up to 10 mg tissue and transfer to a 1.5 mL tubes (one for each sample).
(Cut each 10 mg sample with sterilized cutters as small as possible)
Add 250 μL TL Buffer to each sample.
Add 20 μL Proteinase K Solution to each sample. Vortex to mix.
Incubate at 55°C overnight in a shaking water bath.
Centrifuge at maximum speed (≥4,000 x g) for 5 minutes to pellet undigested tissue
debris and transfer 200 µL of the supernatant to the VWR 96-Well Deep Well Plate (1000 μL) without
disturbing the undigested pellet
List of materials to start to use OT-2
It is necessary to have this equipment to use this pipeline.
Opentrons Equipment List:
| A | B |
|---|---|
| OT-2 | |
| OT-2 8-Channel Pipette P300 | |
| OT-2 Magnetic Module GEN2 | Slot 7 |
Tips & Labware:
| A | B |
|---|---|
| Four Opentrons 200µL Filter Tips | Slot: 5,6,8,9 |
| Two Nest 1-Well Reservoirs (195 mL) | Slot: 10 and 11 |
| VWR 96-Well Deep Well Plate (1000 μL) | Slot 7 on the Magnetic module |
| Two Nest Well plate ( 100 μL) | Slot 2 and 3 |
| Two Nest 12 Well Reservoirs (15 mL) | Slot 1 and 4 |
Star OT-2 run
The pipeline start on the step number 7 of the Mag-Bind® Blood & Tissue DNA HDQ 96 Kit Tissue Protocol
Load the pipeline on the Opentrons app: Extracción.py
The VWR 96-Well Deep Well Plate (1000 μL) labware can be download from: dx.doi.org/10.17504/protocols.io.dm6gp39njvzp/v1
Order of the reagents on the Two Nest 12 Well Reservoirs (15 ml)
Slot 1:
| 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 |
| 11.5 mL of AL buffer | 11.5 mL of AL buffer | Mag-Bind mixed with HBQ buffer (20:340) in a total of 12 mL | Mag-Bind mixed with HBQ buffer (20:340) in a total of 12 mL | Mag-Bind mixed with HBQ buffer (20:340) in a total of 12 mL | 15 ml of VHB buffer | 15 ml of VHB buffer | 15 ml of VHB buffer | 15 ml of VHB buffer |
Slot 4:
| 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 |
| 15 ml of VHB buffer | 15 ml of VHB buffer | 15 ml of VHB buffer | 15 ml of VHB buffer | 15 ml of SPM buffer | 15 ml of SPM buffer | 15 ml of SPM buffer | 15 ml of SPM buffer | Ellution Buffer |
