Solutions:
- Solution A: [Tris HCI 0.1 M (pH 9.0), EDTA 0.1 M, SDS 1%] (keep at RT)
- Phenol-Cloroform: [If done fresh: mix Phenol and Cloroform 1:1, shake, spin for 10 min at 4.000 rpm] (keep at 4°C)
- KAc 8 M (keep at RT)
- Isopropanol (keep at -20°C)
- EtOH 70% (keep at -20°C)
- TE (keep at 4°C)
Procedure:
- Place about 25 flies in an eppendorf tube, keep on ice
- Add 250 μl of solution A
- Homogenize the flies
- Incubate for 30 min at 70 °C
- Add 35μl of KAc
- Shake (no vortexing)
- Incubate for 30 min on ice
- Spin for 15 min at 13.000 rpm
- Move supernatant to a new tube (leave back any precipitate or interphase)
- Add 1 vol of Phenol-Chloroform to (9.) (ca. 250μl)
- Vortex
- Spin for 5 min at 13.000 rpm
- Repeat steps 9 to 12
- Move upper phase to a new tube
- Add 150μl of Isopropanol
- Shake
- Spin for 5 min at 10.000 rpm
- Suck off supernatant using a pipette, not vacuum to avoid losing pellet
- Wash the pellet with 1 ml 70 % EtOH
- Spin for 5 min at 13.000 rpm
- Dry the pellet 10 min under vacuum or on the bench for little longer. Do not over dry
- Resuspend the pellet in 100-200μl of TE