Stratagene standard protocol 1. Pre-chill a 14-ml BD Falcon polypropylene round-bottom tubes on ice. Preheat NZY+ broth to 42℃.
2. Thaw the cells on ice. When thawed, gently mix and aliquot 100 µl of cells into the pre-chilled tubes.
3. Add 4 µl of the β-ME(β巯基乙醇) to the aliquot of cells.
4. Swirl the tubes gently. Incubate the cells on ice for 10 minutes, swirling gently every 2 minutes.
5. Add 0.1-50 ng of the experimental DNA (or 2 µl of a ligation mixture) to the aliquot of cells.
6. Swirl the tubes gently, then incubate the tubes on ice for 30 minutes.
7. Heat-pulse the tubes in a 42℃ water bath for 30 seconds. The duration of the heat pulse is critical.
8. Incubate the tubes on ice for 2 minutes.
9. Add 0.9 ml of preheated (42℃) NZY+ broth and incubate the tubes at 37℃ for 1 hour with shaking at 225-250 rpm.
10. Plate ≤200 µl of the transformation mixture on LB agar plates containing the appropriate antibiotic (and containing IPTG and X-gal if color screening is desired).
11. Incubate the plates at 37℃ overnight. If performing blue-white color screening, incubate the plates at 37℃ for at least 17 hours to allow color development (color can be enhanced by subsequent incubation of the plates for 2 hours at 4℃).