发信人: netbuggie (中原一点虫), 信区: Biology
标 题: Re: another question.
发信站: The unknown SPACE (Fri Jul 14 00:18:01 2000), 站内信件

For your question of slow growth, i suggest you set some controls. first of
all, clean the bottles, get rid of all possible remaining detergent. make
fresh media yourself, pay attention to the salt concentration/pH. while
autoclave, only 15min. when you culture the cell, never let the volume take
too much of the bottle. make sure there is enough O2 supply. since it harbors
a plasmid that has resistance to two different antiboitics, maybe the growth
pressure make it grow very slowly. so set a control of bacteria with only
the vector, no any insert, and see how the bacteria grow. temperature has
to be checked as well. maybe the long insert and expression of the protein
is harmful to the host itself. what do you try to overexpress?

for the second one, i have never used that. it seems that rifampicin inhibits
transcription of RNA, so the protein comes from already induced RNA. gee, can
not believe this, RNA is not so stable. you just try induction of IPTG for
20min and stop the transcription, and then wait to harvest the protein in 1hr.
i do not think it is wise to do so. better to try to call sigma and find out
the true purpose. btw, it is better to optimise the induction conditions,
such as induce the protein at exponential growth or steady growth state,
concentration of IPTG, induction time, harvest time.




【 在 whoknowit (冰毒) 的大作中提到: 】
: Without almost any biology background, I have a lot of questions to
: bother you guys. //hehe..
: I am trying to induce overexpression of one protein from the transformed
: E.coli BL21(DE3)PlysS. The reference says that 20 min after addition of
: IPTG add rifampicin, and then one more hour incubation, harvest the cells.
: who can tell me what is the purpose of the addition of rifampicin?
: The Sigma catalog says: rifampicin is a potent inhibitor of DNA-dependent
: RNA polymerase of bacterial and chloroplast origin. Low inhibitory effect
: on mammalian RNA polumerase.


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