TITLE: Automated QIAcube DNA Extraction
AUTHORS: Clemens Scherzer

[DESCRIPTION]
This protocol explains the Standard Operating Protocol for automated extraction of DNA using QIAcube.

[BEFORE_START]
Based on 12 Samples.

DNA Q/C GOALS 
  1. Cary Concentration Assay 
        a. 260/280 = 1.8-2.0 
        b. Manual Puragene Extraction: 260 µg /mL (65 µg total) of DNA/subject 
        c. Automated QIAcube Extraction: 125 µg/mL (50 µg total) of DNA/subject 
  2. .7% Agarose Gel Electrophoresis 
        a. Human DNA = 23.13 kb with λ DNA-HindIII digest (NEB)

[GUIDELINES]
FREEZER STORAGE
  
Freezers are divided into 4 shelves, with 6 racks per shelf, and 24 boxes that can be held in each shelf. In total, 576 boxes, approximately 2,160 sample sets, can be stored in one -80°C freezer. The first three shelves are designated by visit number: Shelves A1-6 (top shelf) house samples from enrollment visits, shelves B1-6 (2nd shelf) house samples from the 1st year follow-up, and shelves C1-6 (3rd shelf) house samples from the 2nd year follow-up. Shelves D1-6 contain packed red blood cell tubes (PRBC), DNA, and RNA, extracted from blood as described in the protocols above. CSF is designated between two freezers in selected racks. Freezer storage and transactions of samples are recorded in the Freezerworks Inventory software.

[STEPS]
SECTION: Part A: First Elution
1. Heat water bath to 37 °C before starting.

SECTION: Part A: First Elution
2. Thaw Buffy Coat samples by gently agitating sample in water bath.
SECTION: Part A: First Elution
3. Turn on QIAcube. Open door and remove reagent tray from QIAcube. Unscrew all caps and refill reagents where necessary. Do not exceed fill line shown on side of reagent bottle. Make sure reagents are in the proper position in reagent tray.
        a. Position 1: Empty 
        b. Position 2: Buffer AL 
        c. Position 3: 96-100% Ethanol 
        d. Position 4: Buffer AW1 
        e. Position 5: Buffer AW2 
        f. Position 6: Buffer AE
SECTION: Part A: First Elution
4. Add a fresh set of disposable 1000 µL and 200 µL tips to QIAcube.
SECTION: Part A: First Elution
5. Lay out rotor adaptors on holding tray labeled DNA.
SECTION: Part A: First Elution
6. Set up rotor adaptor by placing following materials in the proper positions:
        a. Position 1: QIAamp spin column
                i. Lid Position: L1
        b. Position 2: Empty 
                i. Lid Position: Empty
        c. Position 3: 1.5 mL Eppendorf tube 
                i. Lid Position: L3
 

SECTION: Part A: First Elution
7. Load rotor adaptors in centrifuge of QIAcube.*
 

SECTION: Part A: First Elution
8. Aliquot 200 µL to Sample Tubes RB taking care to pipette up as much Buffy Coat as possible.

SECTION: Part A: First Elution
9. Load Sample Tubes RB into sample tray of QIAcube.*
 

SECTION: Part A: First Elution
10. Aliquot 284 µL into a 1.5 mL Eppendorf tube.* Place in microcentrifuge tube slot A in QIAcube.
 


SECTION: Part A: First Elution
11. Close door of QIAcube and begin program: 
    DNA > QIAamp DNA Blood Mini > blood or body fluid > DNA isolation Part A
SECTION: Part B: Second Elution
12. Remove rotor adaptors and place on sample holding tray.
 

SECTION: Part B: Second Elution
13. Move QIAamp spin column from 1.5 mL Eppendorf tube to Position 1 with lid in Position L1. (Part A step 6)
SECTION: Part B: Second Elution
14. Remove and cap 1.5 mL tube with DNA sample and label with Freezerbondz label DNA-01.
SECTION: Part B: Second Elution
15. Add a fresh 1.5 mL tube to rotor adaptor in Position 3 with lid in Position L3. (Part A step 6)
SECTION: Part B: Second Elution
16. Place rotor adaptors back in the corresponding numbered position in QIAcube’s centrifuge.
SECTION: Part B: Second Elution
17. Add a fresh set of 200 µL disposable tips.
SECTION: Part B: Second Elution
18. Close QIAcube lid and begin program: 
    DNA > QIAamp DNA Blood Mini > blood or body fluid > Add Elution Part B
SECTION: Part B: Second Elution
19. When program has completed, discard QIAamp spin column and remove 1.5 mL tube. Label tube with Freezerbondz label DNA-02.
SECTION: Part B: Second Elution
20. Combine volume of DNA-02 to DNA-01 and pipette up and down to mix to ensure a homogenous mixture of DNA.
SECTION: Part B: Second Elution
21. Split DNA back into two 200 µL.

SECTION: Part B: Second Elution
22. Aliquot 3 µL into a 1.5 mL tube for Cary concentration assay. (Store sample in -20 °C if not being immediately assayed.)

SECTION: Part B: Second Elution
23. Aliquot 2 µL into a PCR tube for .7% agarose gel electrophoresis to confirm for the presence and size of the DNA. (Store sample in -20 °C if not being immediately assayed.)