作者Ren (wisc)
看板Biotech
標題Re: [請益]請問如何表達toxic protein在BL21(DE3)ꤠ…
時間Mon Aug 20 02:08:33 2007
※ 引述《Ren (wisc)》之銘言：
: ※ 引述《qumai (._.)》之銘言：
: : 借用一下文章
: : 剛好也有這樣的問題^^a
: : 請問是一開始就加入2g α-D-Glucose/Glc
: : 然後養到OD>1.0嗎
: Yes.
再補充一下好了，免得說得大家不敢用。
其實一開始還蠻簡單的，就是要refine condition的時候變因會比較多，
最後要fix condition得試比較多條件。
day1: fresh transformation with SOC recovery for 1 hour. (1700-1830)
fresh LB plate with specific antibiotics.
(e.g. BL21(DE3)pLysS with pET system, for Chloramphenicol+AMP)
day2: (+12~ 14 hours in 37C incubator)→
plate ready (7~9:00AM)
（Don't use plate trnasfered for more than 3 days.)
Inoculate 2~5mL LB in the test tube for 6~8hours
(recommend to use multiple colonies for BL21(DE3)pLysS
for the first time )
with shaking ~ 250rpm, 37C (9:00AM to 18:00)→
LB culture ready
Spin down with <2,000rpm slow speed for several minutes
(change LB into M9)
day2 O/N into 250mL flask with 20~50mL M9 (Glc2g)
M9 recipe: M9 salts, NH4Cl, vitamin, Glc 2g, MgSO4, CaCl2
(1800 to 24:00)→ test OD_600nm for OD_max in 20mL M9,→
M9 ready
（Spin down or not)（24:00 to tomorrow 10:00AM)→20mL into 1L.
day3 (10:00AM)→
M9 1L ready→ Add IPTG and 10mL 20%Glc. (Glc2g).
shake another 3~6 even 10 hours. and then harvest.
一開始可以作到nearly no check point. 不過建議你還是在M9的最後階段測一下
OD, 如果你的culture OD<1.5, then your system is much more picky than mine.
我的可以達到3以上。
Good luck!
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◆ From: 76.201.146.194
1F：→ Ren:有問題再問我吧！ 08/20 03:47
2F：推 qumai:真的很感謝您熱心回答 <(_ _)> 08/20 19:37
3F：推 bioeric:很讚的步驟....感謝.... 08/26 03:38