DNA Extraction Qiagen DNeasy

DNA Extraction – Qiagen DNeasy kit.

1. Place sample in 2 mL tube and remove ethanol.
2. Add 180 µL prewarm (56 C )Buffer ATL and 20.uL Proteinase K, vortex 15 sec and spin.
3. Place in the 56 C incubator for 3 hours.
4. Remove from incubator, spin, add 200 µL prewarm (56-70 C)Buffer AL, vortex 15 sec and spin.
5. Place in heat block at 70 C for 10 minutes,spin briefly.
6. Add 200 µL 100% Ethanol, vortex 15 sec and spin . Let stand at room temperature for 20-30 minutes.     Then transfer entire volume (600 µL) onto spin column.
7. Centrifuge at 8000 rpm for 1 minute; discard flow-through.
8. Add 500 µL Buffer AW1 and centrifuge at 8000 rpm for 1 minute; discard flow-through.
9. Add 500 µL Buffer AW2. Centrifuge at 8000 rpm for 1 minutes; discard flow through, then centrifuge at 14,000 rpm for 3 minutes. Discard flow through.
10. Place spin column in 1.5mL tube, add 40 µL buffer AE. Let sit for 5 minutes, then centrifuge at 10000 rpm for 1.5 minute. Keep first elution of 40 µL. Transfer spin column to new 1.5mL tube, repeat with 40 µL buffer AE, let sit for 5 minutes and then centrifuge at 14,000 rpm for 1.5 minute. Keep flow-through as second elution of 40 µL.