介绍： 基 因 型TetrΔ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte[F´proAB lacIqZΔM15 Tn10 (Tetr) Amy Camr]简 要 说 明XL10-Gold是目前转化效率最高的感受态细胞，由Stratagene开发的特异性用于大质粒或珍贵连接产物转化或构建文库的超级感受态细胞。XL10-Gold菌株为Hte(high transformation efficiency)基因型，Hte是Stratagene开发的特异性提高感受态转化效率及大质粒转化能力的宿主菌基因型，已成功应用于40kd质粒的构建。[Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173]赋予XL10-Gold缺失几乎所有已知的限制酶切系统；同时缺失核酸内切酶(endA)，提高了质粒DNA的产量和质量；重组酶缺陷型(recA)减少插入片段的同源重组概率，保证了插入DNA的稳定性；Tetr， Camr赋予菌株四环素和氯霉素抗性；lacIqZΔM15的存在使XL10-Gold可用于蓝、白斑筛选。 High5TM系列XL10-Gold感受态细胞经特殊工艺制作，经pUC19质粒检测转化效率>2×109cfu/μg。 操 作 说 明1. XL10-Gold感受态细胞放置冰中融化（或放手心或室温片刻，待菌体处于冰水混合状态时迅速插入冰中），加入目的DNA（质粒或连接产物）并用手拨打EP管底轻轻混匀，冰上静置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。注 意 事 项1.的感受态细胞最好在冰上融化。2.混入质粒或连接产物时应轻柔操作。3. 转化高浓度的质粒或高效率的连接产物可相应减少最终用于涂板的菌量。4.XL10-Gold菌株对 <40 µg/ml氯霉素有抗性，但对100 µg/ml氯霉素敏感。5.High5TM系列XL10-Gold感受态细胞采用常规转化方法，转化效率可达2×109cfu/μg ；如果有更高要求，可尝试Stratagene公司推荐的标准protocol。Stratagene standard protocol1. 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℃).
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