Purifying DNA from Low-Melt Agarose

from Doug Barrick

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1.† Pour Sea Plaque Agarose Gel (NuSieve) in cold room and allow to set for 1hr.† Use TAE buffer + 0.5 ug/ml EtBr.

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2.† Run gel @ ~70-75V for < 1hr.

3.† Visualize band with long-wave UV light.

4.† Excise gel slice in small volume.

5.† Melt gel slice @68 deg C for 5-15 minutes.

6.† Add 1 volume of TE, 0.6M NaCl† (the final [TE] doesn't matter).† For a 1% gel you want the final solution to be 0.5% agarose and 0.3M NaCl.

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7.† Heat at 65 °C for 2 min.

8.† Add equal volume of phenol/chloroform/isoamyl alcohol.

9.† Vortex at least 30 sec.

10. Plunge in to dry ice for 5 min until frozen.

11. Thaw in water without mixing.

12. Spin @4 deg C for 10 min.

13. Remove aqueous phase. Add equal volume of phenol/chloroform/ isoamyl alcohol.

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14. Vortex at least 30 sec.

15. Spin @4 deg C for 10 min.

16. Remove aqueous phase add to previous extraction.

17. Add glycogen to 5 ug/ml (can use more).

18. EtOH precipitate.† Be careful of loose pellets!!!