Requirements for each sample:
- Elution buffer (0.5 M sodium acetate (pH 7) and 1 mM EDTA (pH 8))
- 95% ethanol
- 80% ethanol
- TE buffer (10x) (pH 8.0)
- Gel fragment containing the DNA
- Excise the region of the gel containing the required DNA band using a scalpel.
- Add elution buffer to the gel slice until the level of the buffer is a few mm above the level of the excised gel band.
- Heat the solution in a water bath at 65ᵒ C until the agarose completely melts.
- Fast-freeze by placing in a -80ᵒ C freezer for 10-15 minutes.
- Immediately thaw the solution by centrifuging for 10 minutes.
- The supernatant is transferred to a new tube.
- Add elution buffer again to the pellet and repeat the steps 3-6.
- Accumulate the supernatants and add an equal volume of 1-butanol.
- Rock the mixture for 15 minutes to remove the EtBr completely from the gel.
- Discard the supernatant and repeat the steps 8 and 9 2-3 times.