本人最近在做蛋白质的诱导表达,我想知道一般的诱导条件是什么?就是诱导时间和加入诱导剂之后的培养时间和条件,以及IPTG的浓度?
一般影响表达的条件是温度,培养基PH值(一般为7.0),IPTG浓度,转速等,常用条件37度,OD600为0.5-1.0间,IPTG浓度我常用0.1mmOL/L,也可以高到1.5mmol/L。转速需要自己摸索,不要高于200r/min。不同的载体和基因都不同。并没有完全适用的万能条件。
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第1个回答 2010-02-02
You have to include IPTG in log growth phase, that is, the OD600 is about 0.5. IPTG concentration can be 0.5 to 2 mM. Sometimes 0.5 mM is not enough, so to be safe, use 1 mM.
Induction time is dependent on what protein you are expressing. You can do a test by collecting samples at induction time 0, 1, 2, 4 hour time points, then run a SDS PAGE to see when is good for your interested protein.
Some protein expression may need lower temperature than 37C. Some use room temperature (25C), some even use lower than that. So you have to test it yourself. In most cases, 37C is OK.
Induction time is dependent on what protein you are expressing. You can do a test by collecting samples at induction time 0, 1, 2, 4 hour time points, then run a SDS PAGE to see when is good for your interested protein.
Some protein expression may need lower temperature than 37C. Some use room temperature (25C), some even use lower than that. So you have to test it yourself. In most cases, 37C is OK.
第2个回答 2010-02-11
IPTG 1/1000 培养到OD0.6后添加,37度诱导4小时