Incubation of transformants - (Jan/16/2006 )
Would somebody tell me what will happen if I keep the transformants (with ampicillin resistance gene in plasmids) incubated in growth medium for several hours before plating onto amp agar plate?
Do I simply get many more colonies on the plate?
Or will there be any disruption in the medium due to the long incubation time?
Why is it common to incubate for something about 1 hour?
Any answer will be helpful.
this is the philosophy as it was given to me:
the 1 hour incubation after transformation is to allow the cells to recover in a nice rich liquid medium before subjecting them to the rigors of plating
the problem with letting them go too long, is that without the antibiotic selection, it is in cell's best interest (metabolically speaking) to kick out the plasmid and grow without it. I dont' know how long this actually takes, and I suspect there is a great deal of variation and it would be difficult to predict. however, if you were to grow your transformants too long, you may find a culture full of healthy happy e coli without any plasmid
on the flip side, adding antibiotic to be able to extend the recovery time is also supposed to be a mistake; you want that brief recovery with no stresses to baby the cells along, ramp up some growth, and get through a population doubling. if you add antibiotic at this step, you might cause a greater mortality rate in the cells. transformation is hard on cells
anyways, this is what I was told back in the day. I hope this helps answer your question?
>the 1 hour incubation after transformation is to allow the cells to recover in a nice rich liquid >medium before subjecting them to the rigors of plating
Recovery.. do you mean the recovery of the cell itself? I thought main point of this step was to allow the cells to express amp resistance gene (or is this included in the word, recovery?)
>the problem with letting them go too long, is that without the antibiotic selection, it is in cell's >best interest (metabolically speaking) to kick out the plasmid and grow without it.
This is very interesting...I did not know that.
Ok...so..if I keep the amp resistant recombinant E.coli in a medium for too long, they would probably be killed by plating onto the amp agar plate, right?
Thank you so much. Your answers help me a lot!
when the cells are recovering, two important things are happening....the are making bla, yeah, but also they are healing; think about what happens during transformation; you are making their cell wall PERMEABLE, then you are shooting DNA inside. this is a pretty harsh process. that is why usually SOC is recommended, and not just LB. SOC is a little richer and therefore the cells don't have to work quite so hard right after the transformation...and yes they have time to make some bla
I would stick to the recommended recovery time...I have seen everything from 30 min to 2 hrs; I usually try to keep it pretty close to an hour myself but a little leeway probably doesn't hurt...although I would not go over 2 hours
I understand about the recovery.
and.. ok.I would stick to an hour incubation anyway.
Thank you for your quick response!
60' recovery should be most optimal. Here what you want is to just allow the bacteira to express the antibiotic resistance gene which it has aquired after transformation. Extending the incubation time may not help in any way but definitely be harmful where few plasmids tend to undergo transformation.