Phenol-based Method for the Isolation of DNA Fragments from Low-Melting Temperature Agarose

Phenol-based Method for the Isolation of DNA Fragments from Low-Melting Temperature Agarose

• Reference: Favre, D. 1992. Biotechniques vol. 13

1. Cut out slice containing DNA, smallest size possible.

    

2. Estimate volume and double with TE (10 mM Tris-HCl, pH 8.0/1 mM EDTA). Melt at 65 C 5-10 min.

    

3. Add 1 volume Tris-buffered phenol at room temperature, mix by inversion.

    

4. Spin 3 min at 10-12k rpm and transfer aqueous phase to new tube. Phenol extract again.

    

5. Spin as in (4) and transfer aqueous phase to new tube containing 0.1 volume 4 M LiCl. Mix by inversion; a white precipitate forms immediately. Place tube on ice 2 min. Spin as above, 3 min.

    

6. Transfer to new tube, leaving transparent pellet behind. Add 1 ul carrier (glycogen) and precipitate with 2.5 volumes cold ethanol. Mix, leave at -70 C 5-10 min, and spin as above, 10 min. Wash pellet with 1 ml 70% ethanol, dry under vacuum, and resuspend in 10-20 ul water or TE.