Sep 12, 2022
DNA Extraction
- 1Department of Genetic Epidemiology, University of Regensburg
Protocol Citation: Klaus J Stark, Kira Julia Stanzick 2022. DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvobjw7l4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 19, 2022
Last Modified: September 12, 2022
Protocol Integer ID: 68916
Abstract
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.
Protocol materials
Proteinase K (20 mg/ml)
Step 18
DNA Hydration SolutionQiagen
In 2 steps
RBC Lysis SolutionQiagen
In 3 steps
Proteinase KQiagen
Step 11
Protein Precipitation SolutionQiagen
In 2 steps
Isopropanol
Step 15
Isopropanol
Step 21
ethanol
In 2 steps
Cell Lysis SolutionQiagen
In 2 steps
Blood clot preparation and red blood cell lysis
Blood clot preparation and red blood cell lysis
40m 19s
40m 19s
Frozen blood clots in 15 ml tubes were transferred from ‑20°C to a warming cabinet 55 °C for00:10:00 and thereafter immediately placed On ice
10m
The tube was inverted to loosen the clot. The blood clot was completely poured with 5 mL RBC Lysis SolutionQiagen into the Clotspin Basket placed on a 50 ml tube
To disperse the clot, centrifugate2000 x g, 00:05:00
5m
The remaining clot material from the Clotspin basket was transferred through the basket to the filtrate with 00:10:00 RBC Lysis SolutionQiagen and the basket was discarded.
10m
To completely disperse the clotted material, the filtrate was vortexed vigorously for00:00:03 and placed for 00:05:00 at Room temperature on a circulating shaker (250 1/min).
5m 3s
The tubes were again vortexed vigorously for 00:00:03 and centrifugated 2000 x g, 00:05:00 at 2000 x g for 5 min
5m 3s
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
The tube was vortexed rigorously for 00:00:10 and additional 5 mL RBC Lysis SolutionQiagen was added to the pellet, followed by vortexing for 00:00:03 and incubation on a circulating shaker (250 1/min) for 00:05:00 at Room temperature
5m 13s
White blood cell lysis
White blood cell lysis
1d 0h 5m 30s
1d 0h 5m 30s
Centrifugation 2000 x g, 00:05:00 to pellet the DNA-containing white blood cells
5m
Discard supernatant carefully, leaving about 0.2 ml of residual liquid. Vortex rigorously for 00:00:10
10s
Adding of 5 mL Cell Lysis SolutionQiagen and 25 µL of Proteinase KQiagen 20 mg/mL as followed by rigorously vortexing for00:00:10
10s
For complete lysis of DNA containing cells, incubate Overnight at 55 °C
10s
Protein precipitation
Protein precipitation
17m 20s
17m 20s
samples were cooled down On ice for 00:05:00 and 1.7 mL Protein Precipitation SolutionQiagen was added, followed by rigorous vortexing for 00:00:20
5m 20s
After centrifugation 2000 x g, 00:10:00 , incubate the samples for 00:02:00 On ice .
12m
DNA precipitation
DNA precipitation
3m
3m
For precipitation of DNA, the supernatant was carefully transferred in a 50 ml tube, containing 5 mL Isopropanol
The samples were mixed by gently inverting the tube 50 times and centrifugated 2000 x g, 00:03:00
3m
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
big red to brown pellet9 steps
Additional washing step to remove remaining erythrocytes
DNA washing
DNA washing
4h 23m 20s
4h 23m 20s
2 mL of Cell Lysis SolutionQiagen and 10 µL of Proteinase K (20 mg/ml)Contributed by users were added, followed by incubation at 55 °C for02:00:00 in a warming cabinet or at Room temperature Overnight on a circulating shaker (250 1/min)
4h
samples were cooled down On ice for 00:05:00 and 1 mL Protein Precipitation SolutionQiagen was added, followed by rigorous vortexing for 00:00:20
5m 20s
After centrifugation 2000 x g, 00:10:00 , the samples were incubated for 00:02:00 On ice
12m
DNA precipitation was done with 3 mL IsopropanolContributed by users . The samples were mixed by gently inverting the tube 50 times and centrifugated 2000 x g, 00:03:00
3m
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
5 mL of 70 % (v/v) ethanolContributed by users were added immediately and the tubes were inverted until the pellet was detached
After centrifugation 2000 x g, 00:03:00 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
3m
DNA hydration
DNA hydration
1h 2m
1h 2m
Addition of 0.5 mL DNA Hydration SolutionQiagen for a large pellet or 0.3 mL for a smaller pellet was followed by incubation at 65 °C in a warming cabinet for 01:00:00 . To fully dissolve the DNA, the sample were put on a shaker Overnight at Room temperature
1h 2m
Samples were centrifugated briefly and the solved DNA was transferred to a 2 ml cup and stored at -20 °C .