>Howdy,
>>I'm working with JM109 as a host cell with pKRT2 as the plasmid. pKRT2=20
>has a _lac_ type operon that is inducible by IPTG. The plasmid also
>affords the host ampicillin resistance. I'm growing the bacteria up in 2x
>YT to an OD of 2.5-2.75 where I add IPTG to a final concentration of 0.5
The rule is that you have to add the IPTG in the middle log phase of
bacterial growth, usually it is 0.4-1.0(O.D), or 1.2, the latest. Almost all
the proteins including the one which you want to express are the products of
primary metabolism, not secondary metabolism, that is why you can not add
IPTG too late.
Basically protein expression is still unpredictable, but we normally know
membrane proteins, especially transmembrane proteins, give troubles, usually
cells stop growing, that is because your protein is uncomapctable=
completely.
>mM. However, even after waiting 6 hours, SDS-PAGE reveals no significant
>difference from uninduced lysate. =20
For expression of a compactable protein there is no big matter for the
concentration of IPTG, but you have to optimise by experiments. As I said
protein is primary metabolism, it usually show products at 2-3hours best
after induction, it gets worse after 16 hours. =20
I hope this help! and you can mail to me directly if you want to know more.
Cheng
>Does anyone have an idea that I could try (the protocol is modified from a
>protocol submitted by D'Aquila and Summers)? Or does anyone have any
>experience with JM109 or could point me to a reference that could tell me
>what OD generally would be late log phase - starting of stationary phase?=
=20
>>>I could just use any sort of help. Thanks.
>>P.S. =20
>>Please respond via e-mail and this newsgroup.
>>Robert T. Love
>Undergrad - Texas A&M University -- Dept. of Chemistry
>rtlove at tamu.edu
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Dr. Cheng Luo Tel. 46 90 165474 =20
Dept. of Plant Physiology Fax 46 90 166676
University of Ume=E5 Email: cheng.luo at plantphys.umu.se
90187 Ume=E5 http://www.plantphys.umu.se/~cheng =20
Sweden =20
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