1. The minced was vortexed and centrifuged then diluted by filtered PBS.
2. 200 µl of sample was taken into 1.5ml micro-centrifuge tubes and 20µl Proteinase K was added.
3. The tubes were vortexed by kept for incubation for 10mins at room temperature.
4. 200 µl of buffer ATL was added to the tubes and mixed well.
5. The samples were incubated in a water bath at 56°C for 1 hour.
6. 200 µl of 100% Et-OH was added to the tubes.
7. The samples were then transferred to the spin columns.
8. The spin columns were centrifuged at 8,000 rpm for 1 min.
9. Collection tubes were changed.
10. 500 µl of AW1 buffer was added to the columns.
11. Again centrifuged at 8,000 rpm for 1 min.
12. The liquid was removed from the collection tubes and placed back.
13. 500 µl of AW2 buffer was added to the columns.
14. Centrifuged at 14,000 rpm for 3 minutes.
15. The columns were placed in a fresh micro-centrifuge tube.
16. 60µl of AE buffer (elution) was added.
17. Centrifuged at 14,000 rpm for 2 minutes.
18. The columns were discarded and the DNA was ready.
using kits (qiagen DNA extracting kits) is the most reliable technique.
I really recommend you not using manual methods. it is just wasting time and energy!
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