EPMotion - DNA Extraction 

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 785µLaliquots 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 565µL aliquots of into 1.5mL tubes

• Store DTT at -20°C
• Note: Note: 1 Aliquot will provide enough reagent for 1 plate.

Aliquot 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)

Note: The following amounts will make a master mix for 96 samples.

Add 15.18mL of to a new 50mL falcon tube.

Add 770µL of Proteinase K to previous 50mL falcon tube

Add 550µL of DTT to previous 50mL falcon tube

Shake vigorously and vortex for 0h 0m 30s

• Place On ice for 0h 3m 0s to allow bubbles to settle

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 150µL 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 900rpm

• Use plates attachment and lid
• Incubate between 12-20 hours

Next Day,

Equilibrate to room temperature for 0h 30m 0s

• Ensure there is at least 25mL of beads in the 50mL falcon tube that you had aliquoted in step 3.

Open epBlue Application.

In the epBlue Studio, open the Application Editor.

Import the epblue protocol Agencourt DNAdvanced Extraction.export7

Open the imported protocol.

• Named as "Agencourt DNAdvanced Extraction"

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 25mL of to reservoir labelled as "Bind 1".

• Place "Bind 1" onto position 1 of the epMotion Reagent Reservoir Rack

Add 25mL of to reservoir labelled as "EB".

• Place "EB" onto position 6 of the epMotion Reagent Reservoir Rack.

Take two clean 50mL falcon tubes and make 100mL of 70% EtOH.

• Add 35mL of to each 50mL falcon tube
• Add 15mL of to each 50ml falcon tube

Add 30mL 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 50mL for 0h 5m 0s.

• Ensure there is no bead pellet collected at the bottom of the tube

Add 25mL of 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

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

Transfer 180µL 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 to blank the Nanodrop.
• Save the nanodrop table only values and assign the file name as the DNA plate code and date.

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.