TITLE: DNA Extraction
AUTHORS: klaus.stark, Kira Julia Stanzick

[DESCRIPTION]
DNA extraction from frozen blood clots is challenging. Here we applied QIAGEN Clotspin Baskets and the Gentra Puregene Blood Kit for DNA extraction from 5.5 ml whole blood without anticoagulating additives. 

[STEPS]
SECTION: Blood clot preparation and red blood cell lysis
1. Frozen blood clots in 15 ml tubes were transferred from ‑20°C to a warming cabinet 55 °Cfor10 min and thereafter immediately placed on ice

SECTION: Blood clot preparation and red blood cell lysis
2. The tube was inverted to loosen the clot. The blood clot was completely poured with 5 mL   into the Clotspin Basket placed on a 50 ml tube

SECTION: Blood clot preparation and red blood cell lysis
3. To disperse the clot, centrifugate2000 x g, 5 min  

SECTION: Blood clot preparation and red blood cell lysis
4. The remaining clot material from the Clotspin basket was transferred through the basket to the filtrate with 10 min  and the basket was discarded. 

SECTION: Blood clot preparation and red blood cell lysis
5. To completely disperse the clotted material, the filtrate was vortexed vigorously for3 s and placed for 5 min at Room temperature on a circulating shaker (250 1/min).
SECTION: Blood clot preparation and red blood cell lysis
6. The tubes were again vortexed vigorously for 3 s and centrifugated 2000 x g, 5 min at 2000 x g for 5 min

SECTION: Blood clot preparation and red blood cell lysis
7. The supernatant was carefully discarded, taking care that the pellet remains in the tube. If no pellet was visible, about 0.5 mL of the supernatant was kept in the tube

SECTION: Blood clot preparation and red blood cell lysis
8. The tube was vortexed rigorously for 10 s and additional 5 mL   was added to the pellet, followed by vortexing for 3 s and incubation on a circulating shaker (250 1/min) for 5 min at Room temperature

SECTION: White blood cell lysis
9. Centrifugation 2000 x g, 5 min  to pellet the DNA-containing white blood cells

SECTION: White blood cell lysis
10. Discard supernatant carefully, leaving about 0.2 ml of residual liquid. Vortex rigorously for 10 s

SECTION: White blood cell lysis
11. Adding of 5 mL   and 25 µL of   20 mg/mL as followed by rigorously vortexing for10 s

SECTION: White blood cell lysis
12. For complete lysis of DNA containing cells,  incubate 10 s at 55 °C

SECTION: Protein precipitation
13. samples were cooled down on ice for 5 minand 1.7 mL  was added, followed by rigorous vortexing for 20 s

SECTION: Protein precipitation
14. After centrifugation 2000 x g, 10 min , incubate the samples for 2 min on ice.

SECTION: DNA precipitation
15. For precipitation of DNA, the supernatant was carefully transferred in a 50 ml tube, containing 5 mL    

SECTION: DNA precipitation
16. The samples were mixed by gently inverting the tube 50 times and centrifugated 2000 x g, 3 min 

SECTION: DNA precipitation
17. The supernatant was carefully discarded, and the tube was drained on a clean piece of absorbent paper without losing the pellet. The DNA pellet was inspected visually and with two following options
SECTION: DNA washing
18. 2 mL of   and 10 µL of   were added, followed by incubation at 55 °C for120 min in a warming cabinet or at Room temperature 120 min on a circulating shaker (250 1/min)

SECTION: DNA washing
19. samples were cooled down on ice for 5 min and 1 mL  was added, followed by rigorous vortexing for 20 s

SECTION: DNA washing
20. After centrifugation 2000 x g, 10 min , the samples were incubated for 2 minon ice

SECTION: DNA washing
21. DNA precipitation was done with 3 mL  . The samples were mixed by gently inverting the tube 50 times and centrifugated 2000 x g, 3 min  

SECTION: DNA washing
22. The supernatant was carefully discarded. The tube was drained on a clean piece of absorbent paper, taking care that the pellet remained in the tube
SECTION: DNA washing
18. 5 mL of 70 % (v/v)   were added immediately and the tubes were inverted until the pellet was detached

SECTION: DNA washing
19. After centrifugation 2000 x g, 3 min  at 2000 x g for 3 min, the supernatant was carefully discarded, and the DNA pellet was air dried at room temperature for 10 min or until the pellet got glassy

SECTION: DNA washing
23. 5 mL of 70 % (v/v)   were added immediately and the tubes were inverted until the pellet was detached

SECTION: DNA washing
24. After centrifugation 2000 x g, 3 min  at 2000 x g for 3 min, the supernatant was carefully discarded, and the DNA pellet was air dried at room temperature for 10 min or until the pellet got glassy
SECTION: DNA hydration
20. Addition of 0.5 mL  for a large pellet or 0.3 mL for a smaller pellet was followed by incubation at 65 °C in a warming cabinet for 60 min. To fully dissolve the DNA, the sample were put on a shaker 2 min at Room temperature

SECTION: DNA hydration
21. Samples were centrifugated briefly and the solved DNA was transferred to a 2 ml cup and stored at -20 °C. 

SECTION: DNA hydration
25. Addition of 0.5 mL  for a large pellet or 0.3 mL for a smaller pellet was followed by incubation at 65 °C in a warming cabinet for 60 min. To fully dissolve the DNA, the sample were put on a shaker 2 min at Room temperature
SECTION: DNA hydration
26. Samples were centrifugated briefly and the solved DNA was transferred to a 2 ml cup and stored at -20 °C.