Re: [問題] 請問有人利用TSS製備competent cell嗎!?
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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