產品介紹

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]簡 要 說 明XL10-Gold是目前轉化效率最高的感受態細胞，由Stratagene開發的特異性用于大質粒或珍貴連接產物轉化或構建文庫的超級感受態細胞。XL10-Gold菌株為Hte(high transformation efficiency)基因型，Hte是Stratagene開發的特異性提高感受態轉化效率及大質粒轉化能力的宿主菌基因型，已成功應用于40kd質粒的構建。[Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173]賦予XL10-Gold缺失幾乎所有已知的限制酶切系統；同時缺失核酸內切酶(endA)，提高了質粒DNA的產量和質量；重組酶缺陷型(recA)減少插入片段的同源重組概率，保證了插入DNA的穩定性；Tetr， Camr賦予菌株四環素和氯霉素抗性；lacIqZΔM15的存在使XL10-Gold可用于藍、白斑篩選。 High5TM系列XL10-Gold感受態細胞經特殊工藝制作，經pUC19質粒檢測轉化效率>2×109cfu/μg。 操 作 說 明1. XL10-Gold感受態細胞放置冰中融化（或放手心或室溫片刻，待菌體處于冰水混合狀態時迅速插入冰中），加入目的DNA（質粒或連接產物）并用手撥打EP管底輕輕混勻，冰上靜置25分鐘。2.42℃水浴熱激35秒（非常重要——Efficiency decreases sharply when cells are heat-pulsed for <30 seconds or for >40 seconds.），迅速放回冰上并靜置2分鐘，晃動會降低轉化效率。3.向離心管中加入700μl不含抗生素的無菌培養基（2YT或LB），混勻后37℃，200rpm復蘇60分鐘。4. 5000rpm離心一分鐘收菌，留取100μl左右上清輕輕吹打重懸菌塊并涂布到含相應抗生素的2YT或LB培養基上。5.將平板倒置放于37℃培養箱過夜培養。注 意 事 項1.的感受態細胞最好在冰上融化。2.混入質粒或連接產物時應輕柔操作。3. 轉化高濃度的質粒或高效率的連接產物可相應減少最終用于涂板的菌量。4.XL10-Gold菌株對 <40 µg/ml氯霉素有抗性，但對100 µg/ml氯霉素敏感。5.High5TM系列XL10-Gold感受態細胞采用常規轉化方法，轉化效率可達2×109cfu/μg ；如果有更高要求，可嘗試Stratagene公司推薦的標準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巰基乙醇) 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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