i prepared pre-culture 20 ml in 3 flasks and added ampicilline and chloramphenicol as antibiotics and etracycline and L-arabinose a inducers
growing up was good during 2 hours and half but when i started the mail culture in 9 flasks ..no growing up after 4 hours i wonder about the suitable volume of pre-culture? it should be 100 ml or it doesn't matter?
Firstly , thank you for your answer ...my main culture should be 1 L *9 flasks and my O.D value 0.6
my pre-culture grown well in 2 hours and half and then transfer it to the main culture but after 4 hours and half the O.D value was too low 0.005
and also i have another question storage of L-arabinose should be at refrigerator or room temp ???
But my professor told me that overnight culture is not good ..i don't know why although my colleagues use this way in their experiments (overnight culture) and it is successfully .. and I need to know about the suitable pre-culture volume should be 100 ml in 1 flask or it is preferable to use it in divided amount ???
hello everybody,
can anyone help me to solve my problem in protein expression I prepared pre-culture in 20 ml LB media using 20 micro gram/ml chloramphenicol, 50 micro gram ampicillin, 5 ng/ ml tetracycline and 0.5mg/ml L-arabinose using glycerol stock . incubate at 37 c and 200 rpm I started from 9: 40 am for 4 hours no growing up of the cells but from many days I prepared pre-culture with the same concentration of antibiotics using colonies from agar plate it grown in 2 hours and half.but when I transfer it to the main culture the O.D value was too low after 5 hours 0.005 what should I do in this problem ?? please help me
The pre-culture might be the problem. Setting up overnight culture is what we normally do. Then dilute the o/n OD600 to 0.15 with your main culture volume the next day. You can then grow the main culture to OD of 0.6 (takes approx. 2 hours) and induce the protein expression for 3-4 hours. Growing up the preculture for 4 hours is not sufficient enough. This should help.
Thank you a lot for your answer and your interest .but my professor told me that overnight culture produce some toxins and it is not efficient .also, when I used cell stock it has no growing for 5 hours. anyway I will try your way (overnight culture )and see the result .
Hi Asmaa Halim,
If you are growing the cells from glycerol stocks then 4 hrs is definitely not enough specially when you are giving so much stress to the cells in the form of antibiotics and all. If you cannot keep the cells for overnight incubation then at least grown the preculture or pre inoculum till OD 1.0.
when you are growing culture from agar plate, the cells are already active as compared to that of the glycerol stocks so they will surely grow faster as you have seen. so for pre inoculum at least have a minimum OD of 1.0 so that it does not get too diluted when you are going for the large scale expression.
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