1. The peripheral blood sample was collected in EDTA vacutainer, 2 ml of which was transferred into a 2 ml tube.
2. Sample was centrifuged for 10 min at 6000 rpm at 4 °C.
3. Plasma was aspirated without touching the leukocyte layer.
4. One ml of RLB was added to the precipitate and mixed gently.
5. The sample was centrifuged for 5 min at 3000 rpm.
6. The supernatant was removed.
Note: If red blood cells still remain, resuspend the cellular pellet in 1 ml of RLB and repeat steps 4-6 for 3 or 4 times until only the white pellet appears.
7. One ml of WLB was added and mixed with white blood cells.
8. The sample was incubated at 65 °C for 30 min.
9. It was centrifuged at 12,000 rpm for 5 min and supernatant was transferred to a new clean tube and pellet was discarded.
10. An equal volume of Chloroform-Isoamilalcohol solution was added to supernatant.
11. The tube was centrifuged at 12000 rpm for 8 min and supernatant was transferred to a new tube.
12. An equal volume of chilled Isopropanol solution was added.
13. Sample was kept in -20 °C for 30 min.
14. Then tube was centrifuged at 4 °C, 12000 rpm for 10 min.
15. The supernatant was discarded and 300 μl of chilled 90% Ethanol was added. Tube was centrifuged at 4 °C, 12000 rpm for 5 min.
16. The 15 and 16 steps were repeated with chilled 70% Ethanol.
17. Supernatant was discarded and pellet was let to be dried at room temperature.
18. Pellet was dissolved in 100 μl of TE buffer or ddH2O and DNA solution was stored at -20 °C
Notification:
1. For frozen blood samples the procedure started from step 4.
2. For clotted blood samples the procedure started from step 7 by adding1 ml of WLB and incubating at 65 °C for 1 hour (shaken every 10 min). Then it was continued by step10.
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