- After inactivating the ligase reaction, cool on ice.
- If enzyme requires high salt adjust salt concentration to the appropriate level with 5 M NaCl.
- Add restriction enzyme (1/2-1 unit/pmol of linker).
- Adjust with H20 to 30 μl.
- Incubate at the appropriate temperature for the restriction endonuclease for 4 hours.
- Extract digestion mixture once with phenol: chloroform:isoamylalcohol (24:23:1) and once more with isoamylalcohol:chloroform (1:24).