why overnight culture is necessary - (Apr/15/2006 )
why overnight culture is necessary for overexpression
i wud put the principle behind the O/N culture in this way, if u take primary innoculum either from agar plates or from glycerol stocks which has been stored for quite some time.
in this case, ur overninght culture allow bacteria to restart the cell metabolism. during this 12 hours time they multiply based on the amount of nutrients present in the medium and damaged cells will die.
at the end they would be in perfect stage, where they can resopond to IPTG stimulation and produce overexpression.
does it make sense to u
overexpression of protein should be done with a great number of bacterias in exponential phase.
When seeding a 1L culture with a single colony, there would be a great time for reaching the 0.3OD
Moreover, the medium would not have the quality required. I mean, bacterias growing are consuming nutriments, and from 1colony to a 0.3OD, most of nutriments of the medium would have been consumed. that would drive a bad growing in general, and the bacterias would express very bad our protein, and more bad if te protein is toxic or grow-disturbing.
So by doing an overnight culture, you are able to seed your big culture by a greater number of bacterias. finally, the seeding culture brings bacterias that are already in suspension, and they are quicker to redo exponential growth than a plated colony.
yeah its good i understand it. its beautiful explanation. u are very talented and clear cocepts. i am very much dull minded. thanks.