I inoculated Rosetta cells in 3 mL of LB with 3 uL of chloramphenicol for 16 hours and they showed growth (turbidity), then in the same day, I put 250 uL of them in a flask with 20 mL and 20 uL of chloramphenicol and incubated them for 2 hours at 37 C in water bath but the OD didn't increase ( there OD supposed to be 04-0.6 after 2 hours of incubation) ??
I repeated this 2 times and I got the same results !!
Rosetta are genetically designed to grow to achieve large biomass. As you have mention OD is not increasing and this might be some wrong setup in your growth media or incubation conditions, I will ask to go for prescribed media and growth conditions.
the incubation was in 37 C water bath with shaking of course.
All the conditions were the same as the protocol that the doctor gave me.
I didn't measure the starting OD.. but after I incubated them for extra 3 hours (total time = 5 hours), the OD increased.. but we need the cells to be in the exponential phase before we make them competent, I hope the extra hours of incubation didn't affect the phase stage.
Dear,
I think
1- concentration of chloramphenicol is much more.
2- incubation time is too less than enough.
You should check out incubation time at the first, if you can't obtain the favorite result you should decrease concentration of chloramphenicol.
what it seems to be happen is that cell in the preincubation period arrived to late stationary phase. So, when you dilute them in fresh media it takes a long lag period, before to start growing. Do not worry, just control your OD of the culture and when it reach 0.6, just collect the cell and make them competent.
I think that what Dr. Juan said is what really happened in my case, because I thought the preincubation period should be 16 hours for Rosetta cells.. which made them reach stationary and the OD changed slowly...maybe it should be less.
Thank you so much !
The cells could be still in lag phase after 2 hours using a 3ml 16h innocum broth culture. Switching media from LB to a less rich media will lead to a longer lag phase too..
Dear Sarah Daib
I think you already sorted out your problem but I was thinking to share my own experience as other may learn from it.
I encountered similar problem as you. Transformed four different constructs (same vector backbone) in Rosetta II, did an O.N pre-inoculum (50 mL, in 2xYTmedia, amp/chloramph), add this in 1 L 2xYT media. The cultures did not grow even after 9 hours (37 degree). They simply stayed at the same OD as when I started the cultures (OD ~ 1.6-1.9).
Afterwards from the same plate did another O.N pre-inoculum (same conditions as the first time) but when I inoculated the large cultures I adjusted the OD to 0.1. In two hours all four cultures reached 0.5-0.6.
Therefore my opinion is that one should pay more attention to the OD when starting the culture for expression.
Dave Siak Wei Ow yes switching media leads to longer lag phase in every cell case but RosettaDE3 strain takes more time i too could not find the exact reason. can u plz be specific to it because expressiing in BL21 does not takes that much time as Rosetta DE3 is taking.
Thanks.
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