TITLE: Manual QIAgen DNA Extraction
AUTHORS: Clemens Scherzer, Bradley Hyman, Charles Jennings

[DESCRIPTION]
This protocol explains the Standard Operating Protocol for manually extracting DNA using Qiagen.

[BEFORE_START]
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: Manual Qiagen DNA Extraction
1. Thaw buffy coats or whole bloods by equilibrating to Room temperature.

SECTION: Manual Qiagen DNA Extraction
2. Heat a heating block to 56 °C.

SECTION: Manual Qiagen DNA Extraction
3. Gather all the necessary reagents/buffers (Buffer AE, Buffer AW1, Buffer AW2, Buffer AL, 100% ethanol, Qiagen Protease).
SECTION: Manual Qiagen DNA Extraction
4. If a precipitate has formed in Buffer AL, dissolve by incubating in 56 °C.

SECTION: Manual Qiagen DNA Extraction
5. Gather appropriate number of QIAamp Mini spin columns and 2ml collection tubes.
SECTION: Manual Qiagen DNA Extraction
6. Pipet 20 µL into the bottom of a 1.5ml microcentrifuge tube.

SECTION: Manual Qiagen DNA Extraction
7. Add 200 µL to the 1.5ml microcentrifuge tube.

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8. Add 200 µL to the sample and mix by pulse-vortexing for 15 s at half-speed. 
 


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9. Incubate on 56 °C heat block for 10 min.
 



SECTION: Manual Qiagen DNA Extraction
10. Briefly centrifuge the 1.5ml microcentrifuge tubes to remove drops from the inside of the lid.
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11. Add 200 µL to the sample, and mix again by pulse-vortexing for 15 s. After mixing, briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.

SECTION: Manual Qiagen DNA Extraction
12. Carefully add the sample from step 6 to the QIAamp Mini spin column (in a 2ml collection tube) without wetting the rim. Close the cap, and centrifuge at 20000 x g, 1 min.

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13. Place in a clean 2 mL collection tube.
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14. Carefully open the QIAamp Mini spin column and add 500 µL without wetting the rim. Close the cap and centrifuge at 6000 x g, 1 min. Place the QIAamp Mini spin column in a clean 2ml collection tube and discard the collection tube containing the filtrate.

SECTION: Manual Qiagen DNA Extraction
15. Carefully open the QIAamp Mini spin column and add 500 µL without wetting the rim. Close the cap and centrifuge at 20000 x g, 3 min.

SECTION: Manual Qiagen DNA Extraction
16. Place the QIAamp Mini spin column in a new 2ml collection tube and discard the old collection tube with the filtrate. Centrifuge at 20000 x g, 1 min.
 

SECTION: Manual Qiagen DNA Extraction
17. Place the QIAamp Mini spin column in a clean 1.5ml microcentrifuge tube labeled with HBS IDs and discard the collection tube containing the filtrate. Carefully open the QIAamp Mini spin column and add 150 µL. Incubate at 56 Room temperature for 5 min and then centrifuge at 6000 x g, 1 min.

SECTION: Manual Qiagen DNA Extraction
18. Repeat step 11 for a second elution of 150μl of DNA.
SECTION: Manual Qiagen DNA Extraction
19. Combine the two 150μl aliquots and mix and split them.