Removal of Terminal Phosphate Groups

If starting with a restriction digest, heat-inactivate the restriction endo-

nuclease (10 min, 70°C), and then dilute 11 with 2X CIP buffer. If starting with purified DNA in water or IX TE, then also dilute 1:1 with

2X CIP buffer.

After incubation, phosphatases cannot be heat-inactivated, so add 80 pL of IX TE, and then 100 pL of saturated phenol. Vortex well (1 min).

Following centrifugation, remove the upper, aqueous layer, and repeat the extraction twice more.

5. Either directly apply the aqueous layer to a preparative gel (see Chapters 51 and 11 and Note 9), or add 6.5 pL 5M NaCl and 250 pL ice-cold absolute ethanol, incubate at -20°C for 30 min to 1 h, and recover DNA by centrifugation.

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