TITLE: EPMotion - DNA Extraction 
AUTHORS: Khai-Minh H Nguyen, Katarina A Cohen, Oksana Polesskaya, Abraham Palmer

[DESCRIPTION]
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

[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. 

[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). 

[STEPS]
SECTION: Lysis Master Mix
4. Note: The following amounts will make a master mix for 96 samples.

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

SECTION: Agencourt DNAdvance Reagent Preparation
1. 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 µLaliquots 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. 


SECTION: Lysis Master Mix
5. Add 770 µL of Proteinase K to previous 50mL falcon tube 

SECTION: Agencourt DNAdvance Reagent Preparation
2. Create 565 µL aliquots of   into 1.5mL tubes
Store DTT at -20 °C 
Note: Note: 1 Aliquot will provide enough reagent for 1 plate. 


SECTION: Lysis Master Mix
6. Add 550 µL of DTT to previous 50mL falcon tube 

SECTION: Lysis Master Mix
7. Shake vigorously and vortex for 30 s 
Place on ice for 3 min to allow bubbles to settle 


SECTION: Overnight Lysis
9. 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 


SECTION: Overnight Lysis
8. 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


SECTION: Overnight Lysis
10. 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 

SECTION: Overnight Lysis
11. Place deepwell plate on Thermomixer C and incubate overnight at    
Use plates attachment and lid
Incubate between 12-20 hours   


SECTION: Setting up the EPmotion
13. Open epBlue Application. 


SECTION: Setting up the EPmotion
14. In the epBlue Studio, open the Application Editor.

SECTION: Setting up the EPmotion
15. Import the epblue protocol   

SECTION: Setting up the EPmotion
16. Open the imported protocol.
Named as "Agencourt DNAdvanced Extraction"
SECTION: Setting up the EPmotion
17. 
  
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. 

SECTION: Setting up the EPmotion
12. Next Day, 
Equilibrate    to room temperature for 30 min 
Ensure there is at least 25 mL of beads in the 50mL falcon tube that you had aliquoted in step 3. 


SECTION: Agencourt DNAdvance Reagent Preparation
3. 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)



SECTION: Setting up the EPmotion
18. 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" 

SECTION: Setting up the EPmotion
20. Add 25 mL of  to reservoir labelled as "Bind 1". 
Place "Bind 1" onto position 1 of the epMotion Reagent Reservoir Rack 

SECTION: Setting up the EPmotion
21. Add 25 mL of   to reservoir labelled as "EB".
Place "EB" onto position 6 of the epMotion Reagent Reservoir Rack. 


SECTION: Setting up the EPmotion
22. Take two clean 50mL falcon tubes and make 100 mL of 70% EtOH.
 Add 35 mL of   to each 50mL falcon tube
 Add 15 mL of   to each 50ml falcon tube

SECTION: Setting up the EPmotion
23. 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.

SECTION: Setting up the EPmotion
19. Place reservoir labelled as "Empty" onto position 7 of the epMotion Reagent Reservoir Rack  
SECTION: Setting up the EPmotion
24. Shake and Vortex the previously aliquoted 50mL  for 5 min. 
Ensure there is no bead pellet collected at the bottom of the tube


SECTION: Setting up the EPmotion
25. Add 25 mL of   to reservoir labelled as "Beads" 
Place "Beads" onto position 2 of the epMotion Reagent Reservoir Rack


SECTION: Setting up the EPmotion
26. Remove all lids off reagent reservoirs (including "Empty" reservoir) and place reservoir rack onto position C5 of the epMotion worktable. 
SECTION: Setting up the EPmotion
27. 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.  
SECTION: Setting up the EPmotion
28. 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.
SECTION: Setting up the EPmotion
29. Close epMotion hood when all tips/plates/reagents are in position
SECTION: Running the Protocol
30. Press the play button
SECTION: Running the Protocol
31. Select your EPmotion ID and press "Next"
SECTION: Running the Protocol
32. Select the "Input volumes manually" option under the Volume Settings section and press "Next"
SECTION: Running the Protocol
33. 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.

SECTION: Running the Protocol
34. 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
SECTION: Running the Protocol
35. Press "Run" 

SECTION: Running the Protocol
36. 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".
SECTION: Running the Protocol
37. 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   

SECTION: Day After Extraction
38. 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 

SECTION: Day After Extraction
39. Seal, shake and spin down the newly transferred plate. 

SECTION: Day After Extraction
40. 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. 

SECTION: Database Entry
41. Open the Extraction Database that you created in the "Sample Cutting/Processing Protocol".

SECTION: Database Entry
42. Copy and paste the nanodrop concentrations, 260/280, and 260/230 values into the google sheets Extraction Database. (Columns H-J) 

SECTION: Database Entry
43. Enter the date in which you performed the extraction in Column G 
SECTION: Database Entry
44. Enter the plate barcode (as shown below)  into Column B of the Extraction Database.