常规操作方法 1. XL10-Gold感受态细胞从-80℃拿出,迅速插入冰中,5分钟后待菌块融化,加入目的DNA (质粒或连接产物) 并用手拨打EP管底轻轻混匀 (避免用枪吸打),冰中静置25分钟。 2. 42℃水浴热激35秒(非常重要——Efficiency decreases sharply when cells are heat-pulsed for <30 seconds or for >40 seconds.),迅速放回冰上并静置2分钟,晃动会降低转化效率。 3. 向离心管中加入700μl不含抗生素的无菌培养基(2YT或LB),混匀后37℃,200rpm复苏60分钟。 4. 5000rpm离心一分钟收菌,留取100μl左右上清轻轻吹打重悬菌块并涂布到含相应抗生素的2YT或LB培养基上。 5. 将平板倒置放于37℃培养箱过夜培养。
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℃).