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

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

[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 Gentra Puregene DNA Extraction
4. Add 3 mL and mix by inverting 10 times.

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5. Incubate sample for 5 min at 65 Room temperature (15°C-25°C). Invert once during the incubation.

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6. Centrifuge at 2000 x g, 2 min, 25 °C.

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7. Pour off and discard supernatant. Leave approximately 200 µL of residual liquid and the white blood cell pellet.
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8. Vortex tube on high speed to resuspend pellet in the residual liquid.
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9. Add 3 mL. Vortex on high speed for 10 s. 
 

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10. Add 1 mL. Vortex on high speed for 20 s.

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11. Centrifuge sample at 2000 x g, 5 min, 25 °C. 
 

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12. Add 3 mL into a clean 15 mL tube. Carefully pour the supernatant from step 11 into this tube. 
 


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13. Invert samples 50 times until DNA is visible as threads or a clump.
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14. Centrifuge tube at 2000 x g, 3 min. Observe DNA as a small white pellet.

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15. Carefully pour/pipette off supernatant without disturbing the pellet. Drain excess drops of supernatant by inverting on a Kimwipe, while making sure that pellet remains in the tube.
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16. Add 3 mL and invert 3-5 times to wash the DNA pellet.

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17. Centrifuge pellet at 2000 x g, 1 min, 25 °C.

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18. Carefully pour/pipette off ethanol without disturbing the pellet. Drain excess drops of ethanol by inverting on a Kimwipe, while making sure that pellet remains in the tube.
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19. Air dry the pellet for 1 min. 
 


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20. Add 250 µL. Vortex on medium speed for 5 s.

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21. Incubate DNA in 65 °C water bath for 60 min in order to dissolve DNA.

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22. Incubate at 65 Room temperature (25°C) 60 min on rocker.

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23. On the following day, DNA can be transferred to a 1.5 mL tube. Split volume in half to create two aliquots of DNA (DNA0-01 and 02).
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24. Aliquot 3 µL into a 1.5 mL tube for Cary concentration assay. (Store in -20 °C if not being assay immediately.)

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25. Aliquot 1.5 µL into a PCR tube for .7% agarose gel electrophoresis to confirm presence and size of DNA. (Store in -20 °C if not being assay immediately.)
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1. Preheat water bath to 37 °C before removing whole blood samples from -80°C freezer. 
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2. Thaw whole blood samples by gently agitating sample in the 37°C water bath. Once thawed, reset water bath to 65 °C for last step in protocol. 

SECTION: Manual Gentra Puregene DNA Extraction
3. Add 9 mL to a 15 mL centrifuge tube.