Sep 12, 2022
Version 1
EPMotion - DNA Extraction V.1
- 1UC San Diego;
- 2Palmer Lab
Protocol Citation: Khai-Minh H Nguyen, Katarina A Cohen, Oksana Polesskaya, Abraham Palmer 2022. EPMotion - DNA Extraction . protocols.io https://dx.doi.org/10.17504/protocols.io.8epv59reng1b/v1
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: March 17, 2022
Last Modified: September 12, 2022
Protocol Integer ID: 59577
Abstract
This protocol is designed for Agencourt's DNAdvance Extraction kit on the EPmotion 5075. Samples are extracted in a 96 well plate. This is a continuation of the "Sample Cutting/Processing" Protocol
Guidelines
This protocol is a continuation to the Sample Cutting/Processing Protocol . If you do not have an EPmotion, follow the Agencourt DNAdvace protocol (download the handbook from Step 1 of this protocol).
Materials
Note
EPmotion tips are very limited during this pandemic. The tips listed below are tip reloads. You will need to request eppendorf to send a minimum of 8 x 300ul tip trays and 1 x 50ul tip tray.
Equipment
- Eppendorf ThermoMixer C Catalog No. 5382000023
- Eppendorf SmartBlock plates attachment Catalog No. 5363000039
- Eppendorf Lid for Thermomixer Catalog No. 5363000233
- epMotion 5075
- 8 Channel Dispensing Tool (300uL) Catalog No. 960001052
- 8 Channel Dispensing Tool (50uL) Catalog No. 960001044
- Gripper Tower
- Eppendorf Magnum FLX Magnet Adapter Catalog No. 960001044
- Thermoadaptor for PCR Catalog No. 960002199
- Eppendorf Reservoir Rack (up to 7 reservoirs) Catalog No. 960002148
- Centrifuge that can spin down plates
- DynaMag™-96 Side Magnet Catalog No. 12331D
Reagents Supplied by User
- 100% Ethanol 200 Proof
- 1M DTTSigma AldrichCatalog #43816 or equivalent
- Ultrapure Distilled, Nuclease Free Water
Consumables
- 50mL Falcon Tube
- 25mL Reagent Reservoir
- 200uL tips
- 200uL Multichannel Pipette
- 1-50uL epT.I.P.S. Motion Catalog No. 0030014413
- 20-300uL epT.I.P.S. Motion Catalog No. 0030015231
- epMotion reservoir 30mL Catalog No. 960051009
- MicroAmp™ Optical 96-Well Reaction Plate with Barcode Catalog No. 4306737
- Adhesive PCR Plate Seals Catalog AB0558
Protocol materials
100% EtOH
Step 22
Ultrapure Distilled, Nuclease Free Water
Materials, Step 22
1M DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #43816
Materials, Step 2
Lysis BufferBeckman Coulter GenomicsCatalog #C42203
Step 4
Bind BBE (magnetic beads) Beckman Coulter Genomics
In 4 steps
Pre-Bind PBBA Beckman Coulter Genomics
Step 20
Elution EBABeckman Coulter Genomics
In 2 steps
Safety warnings
You will be working with animal tissues.
Before start
This protocol assumes that you have the EPmotion set up and epBlue software downloaded. Complete the "Sample Cutting/Processing Protocol" before you start on this protocol.
Agencourt DNAdvance Reagent Preparation
Agencourt DNAdvance Reagent Preparation
30m
30m
Download the DNAdvance Handbook from Beckman Coulter Genomics. Google this - (PN B66866AC) or request from Beckman Coulter Group.
Prepare Proteinase K Solution following instructions on page 9 of the Handbook.
- Create 785 µL aliquots of proteinase K solution in 1.5mL tubes.
- Store Proteinase K solution at -20 °C
- Note: 1 Aliquot will provide enough reagent for 1 plate.
Create 565 µL aliquots of 1M DTTSigma AldrichCatalog #43816 into 1.5mL tubes
- Store DTT at -20 °C
- Note: Note: 1 Aliquot will provide enough reagent for 1 plate.
Aliquot Bind BBE (magnetic beads) Beckman Coulter Genomics into 50mL falcon tubes for ease of use.
- Ensure you fully vortex and shake the beads (there should be no beads collected at the bottom of the bottle)
Lysis Master Mix
Lysis Master Mix
5m 30s
5m 30s
Note: The following amounts will make a master mix for 96 samples.
Add 15.18 mL of Lysis BufferBeckman Coulter GenomicsCatalog #C42203 to a new 50mL falcon tube.
Add 770 µL of Proteinase K to previous 50mL falcon tube
Add 550 µL of DTT to previous 50mL falcon tube
Shake vigorously and vortex for 00:00:30
- Place On ice for 00:03:00 to allow bubbles to settle
3m 30s
Overnight Lysis
Overnight Lysis
Spin down Deepwell plate with processed samples from the Sample Cutting/Processing Protocol.
- Defrost Deepwell plate with processed samples if stored in -80 °C on ice.
- Spin at 2500rpm for 5 sec
Pour Master Mix into a reagent reservoir and Pipette 150 µL of Master Mix into each well of the deepwell plate.
- Use Multichannel pipette for efficiency
Seal deepwell plate with a Square sealing mat (Cat. AB0675) and quickly spin down on centrifuge
- Ensure all wells are tightly covered.
- Spin at 2500rpm for 5 sec
Place deepwell plate on Thermomixer C and incubate overnight at 900 rpm, 55°C
- Use plates attachment and lid
- Incubate between 12-20 hours Overnight
Setting up the EPmotion
Setting up the EPmotion
30m
30m
Next Day,
Equilibrate Bind BBE (magnetic beads) Beckman Coulter Genomics to room temperature for 00:30:00
- Ensure there is at least 25 mL of beads in the 50mL falcon tube that you had aliquoted in step 3.
30m
Open epBlue Application.
In the epBlue Studio, open the Application Editor.
Import the epblue protocol 
Agencourt DNAdvanced Extraction.export7
Agencourt DNAdvanced Extraction.export7 Open the imported protocol.
- Named as "Agencourt DNAdvanced Extraction"
DNA Extraction Worktable Setup
Set up EPmotion following the worktable figure above.
- Place 300ul EPmotion tip rack on positions A2, A3, B1-B4 without tip rack lid.
- Place 300ul EPmotion tip rack on positions B0 and C4 with tip rack lid.
- Place 50ul EPmotion tip rack on position B5 without tip rack lid.
- Place eppendorf Thermoadapter PCR 96 on position C3
- Place eppendorf magnum FLX magnet on position C2
- Ensure Tip waste basket is empty and liquid waste reservoir is empty.
Labelling epMotion Reservoirs
- Grab 7 epMotion 30mL Reservoir Racks
- Label 1 epMotion Reservoir as "Bind 1"
- Label 1 epMotion Reservoir as "Beads"
- Label 3 epMotion Reservoirs as "70% EtOH"
- Label 1 epMotion Reservoir as "EB"
- Label 1 epMotion Reservoir as "Empty"
Place reservoir labelled as "Empty" onto position 7 of the epMotion Reagent Reservoir Rack
Add 25 mL ofPre-Bind PBBA Beckman Coulter Genomics to reservoir labelled as "Bind 1".
- Place "Bind 1" onto position 1 of the epMotion Reagent Reservoir Rack
Add 25 mL ofElution EBABeckman Coulter Genomics to reservoir labelled as "EB".
- Place "EB" onto position 6 of the epMotion Reagent Reservoir Rack.
Take two clean 50mL falcon tubes and make 100 mL of 70% EtOH.
- Add 35 mL of 100% EtOHContributed by users to each 50mL falcon tube
- Add 15 mL of Ultrapure Distilled, Nuclease Free WaterContributed by users to each 50ml falcon tube
Add 30 mL of 70% EtOH made from the previous step to the 3 resevoirs labelled as "70% EtOH"
- Place the three "70% EtOH" reservoirs onto positions 3, 4, and 5 of the epMotion Reagent Reservoir Rack.
Shake and Vortex the previously aliquoted 50mLBind BBE (magnetic beads) Beckman Coulter Genomics for 00:05:00 .
- Ensure there is no bead pellet collected at the bottom of the tube
5m
Add 25 mL of Bind BBE (magnetic beads) Beckman Coulter Genomics to reservoir labelled as "Beads"
- Place "Beads" onto position 2 of the epMotion Reagent Reservoir Rack
Remove all lids off reagent reservoirs (including "Empty" reservoir) and place reservoir rack onto position C5 of the epMotion worktable.
Remove deepwell plate containing all lysed samples from Thermomixer and spin down.
- Remove mat seal from deepwell plate and place on position C1 of the epMotion worktable.
- Ensure that the plate is placed in the correct orientation. Letters and Numbers should be right side up on the plate.
Take new a MicroAmp™ Optical 96-Well Reaction Plate with Barcode (Catalog No. 4306737) and label with same plate name as the deepwell plate, your initials and today's date.
- Place labelled plate on the thermoadapter on position C3
- Ensure that the plate is placed in the correct orientation. Letters and Numbers should be right side up on the plate.
Close epMotion hood when all tips/plates/reagents are in position
Running the Protocol
Running the Protocol
Press the play button
Select your EPmotion ID and press "Next"
Select the "Input volumes manually" option under the Volume Settings section and press "Next"
Press "Next" again until you see this page below.
When on the page above, select the "Set all volumes" option and enter "200".
- This will enter the volume of lysate in each well of the deepwell plate to 200ul.
'
Press "Next" when all the fields are set to 200ul.
Ensure that the volumes of the reagents are set to:
- Bind 1 - 25000ul
- Bind 2 - 25000ul
- EtOH 1 - 30000ul
- EtOH 2 - 30000ul
- EtOH 3 - 30000ul
- Elution Buffer - 25000ul
Press "Run"
About halfway through the protocol, there will be a "user intervention" step. The robot will stop and ask you to spin down the deepwell plate and return to magnet.
- Take deepwell plate out and pulse-fuge. Return to magnet and Press "OK".
Once the robot is done, open the hood and remove the Elution plate from position C3.
- NOTE: There will be bead carryover to the elution plate.
- Seal elution plate with a PCR adhesive seal
- Place elution plate on DynaMag - 96 Side Magnet
- Place magnet and plate at 4C and leave Overnight
Day After Extraction
Day After Extraction
Transfer 180 µL of supernatant to a new MicroAmp™ Optical 96-Well Reaction Plate with Barcode (Catalog No. 4306737)
- Label the new plate the same as the old plate
- If magnetic beads are not separated from the supernatant, wait for a few more hours
- Try not to carry over beads to the new plate
Seal, shake and spin down the newly transferred plate.
Nanodrop the plate.
- Use the Elution EBABeckman Coulter Genomics to blank the Nanodrop.
- Save the nanodrop table only values and assign the file name as the DNA plate code and date.
Database Entry
Database Entry
Open the Extraction Database that you created in the "Sample Cutting/Processing Protocol".
Copy and paste the nanodrop concentrations, 260/280, and 260/230 values into the google sheets Extraction Database. (Columns H-J)
Enter the date in which you performed the extraction in Column G
Enter the plate barcode (as shown below) into Column B of the Extraction Database.