Restriction digestion

1. Vector: 5 ul low-copy plasmid (100 ng ~ 200 ng) or 1 ul high-copy plasmid (100 ng to 200 ng)

2. PCR product: 5 to 10 ul.

3. Insert from another vector: 200 ng to 500 ng.

4. Add 1.5 ul buffer (FastDigest from Fermentas or NEB).

5. Add water to ~14.5 ul.

6. Add 0.3 ul enzymes (e.g., 0.3 ul EcoR1 and 0.3 ul Xho1).

7. Digest at 37 oC (some enzymes may require up to 50 oC) for 30 min (FastDigest) or 1 to 2 hours (NEB).

8. Purify vector/insert/PCR band by gel electrophoresis and spin column.

9. If donor vector has "dead" selection cassette, heat digestion mix at 75 oC for 15 min, cool, add some water, mix with insert (use 75 oC heat to inactivate restriction enzymes), add ligation buffer and ligase, then ligate at RT for 30 to 2 hour or overnight.