我常用的Protocol: (我大概每3-4個月會作一次) 1.Day1: Incubate at 37度 on LB plate for 12-16hr. (From bacterial stock ; JM109 or DH5alpha) 2.Day2: Steak a single colony into 5ml of LB medium, then incubate at 37度 for 12-16hr. 3.Day3: Transfer 3ml of overnight culture into 200ml of LB, then incubate at 37度 for addition 3hr or so.(OD600=0.375-0.4) 4.Centrifuge 2500 rpm at 4度 for 10min, then discard the supernatant. 5.Add 100ml of ice-cold 50mM CaCl2 (autoclaved) to cell pellet and re-suspend cells completely. Place on ice for 30min. 6.Centrifuge 2500rpm at 4度 for 10min, then discard the supernatant. 7.Add 12ml of ice-cold TSS with 5% DMSO to cell pellet and re-suspend cells completely. 8.Make 200ul of aliquots and put on ice for 30min. Then, using liquid N2 to freeze cell. 9.Use 100ul of competent cells to perform transformation. TSS: (Autoclaved; fresh prepared) Stock final 150ml PEG3350 10% 15g MgSO4 1M 25mM 3.75ml MgCl2 1M 25mM 3.75ml Tryptone 1.5g Yeast ext. 0.75g NaCl 0.75g ( 12ml TSS 中加入 600ul 原倍DMSO ) ※ 引述《koele1984 (koele1984)》之銘言： : 請問有人利用TSS(Transformation and Storage Solution : for chemical transformation)來製備competent cell嗎!? : 我找到了2份的protocol : solution的配法大同小異 : 只不過我在test efficiency時 效果很差 : 我想有幾個原因 : 1.我當初在加PEG的時候，經過autoclave後仍有一些沒溶解 : 2.有些protocol加完TSS後不用放在冰上20分鐘，有些有要求 : (我加完後就直接拿去-80凍了) : 目前找到的就這幾個問題 : 請幫我看看.....Orz : ps.我可以順便要TSS的protocol嗎!? : 我想比較看看哪個比較好.... --

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