- Hold competent cells (from -80℃ refrigerator) on ice.
- Gently mix ligation product (10-15 μL) with cells, then bath in ice for 30 min.
- (Precisely!) Bath in 42℃ water for 90 sec.
- Hold again in ice for 2 min.
- Bath in 37℃ for 45 min. For plasmid pKO3 (and other temperature-sensitive strains), bath in 30℃ for 1 hour.
- Separate cells on petri-dishes, and cultivate them in 37℃ for 12 hours (for pKO3, 24 hours are necessary).