Ligation

1. Digest plasmid or purified PCR product with 0.3~0.5 ul Fermentas or NEB enzymes with Fermentas FastDigest Buffer (green) or NEB Buffer.

Plasmid (e.g., vector): 37 oC for 30min to 1 hour.

Purified PCR product: 37 oC for 1~2 hours, sometimes overnight

2. Run agarose gel to purify vector and insert.

3. Cut out vector and PCR bands

4. Dissolve gel in QG gel-solubilization buffer at 55 ~65 oC. Mix insert (or digested PCR product) with vector.

5. Purify DNA using spin column: load both dissolved DNA solutions to a column, spin for 30 sec, then wash 2X 350 ul of PE (spin for 15 sec at max speed), then spin 1 min at mix speed.

6. Elute DNA using 20 ul autoclaved ddH2O.

7. Add 2.5 ul ligation buffer and 1 ul ligase.

8. Ligate at RT for 30 min to 2 hours, or overnight.

9. Use entire ligation reaction for transformation.