Hi all,
i am currently using rosetta competent cell to expressing my target protein, I incubated to OD~0.4, using IPTG induction in 25C overnight, however the next day OD only ~0.5, it did not happened in previous attempts.
Any clue?
Hi Tang Zhi Zhuang,
If I may know, how much the concentration of IPTG did you put on the culture? and also which vector did you use for expressing your target protein?
Because sometimes you need to do some trial and error to get better condition for expression the protein. For example: one of my protein will be good at expression using IPTG 0.1 M, while others will need more.
and also I did induction only for 2-3 hours at 37oC, I will get the OD ~0.6-0.8
As far as I know, for the induction, usually will do the incubation at 37oC
Best,
-Fean-
Hello,
I have done IPTG inductions quite a few times and I also did the majority of them at 25oC. Does your procedure call for the addition of new media once you add IPTG? The bacteria may have exhausted the media causing a slow growth rate. Usually we added 25% more media at the start of the IPTG induction.
Also, why are you concerned about the OD of your cells after adding IPTG? Why not just do a time course (0 hour, 4 hour, 8 hour, o/n etc..) and analyze it on SDS-PAGE? That will give you more information about protein expression than the OD will.
Best,
David
- Badges
- Science method