Do you grow bacteria at 37°Cafter heat shock, before plating? - (Feb/07/2007 )
Hi there!
I have a question.
Do you guys grow your bacteria in LB or SOc medium wo antibiotics after you heat shock them (before plating). I remember my old supervisor saying that this was not required for amp. resist plasmids.But I do not remember why?
Would be nice to hear why and what you do.
Thanks
I have a question.
Do you guys grow your bacteria in LB or SOc medium wo antibiotics after you heat shock them (before plating). I remember my old supervisor saying that this was not required for amp. resist plasmids.But I do not remember why?
Would be nice to hear why and what you do.
Thanks
I do this everytime with every plasmid. I think that the bacteria need some "rest" after the heat shock to recover.
Definitively the same.
After heat shock, cells need a recuperation period for recovery (elevated temperature causes membrane to move around and the holes get bigger). LB or SOC helps to get the cells healthy (“makes the cells happy” said someone 
). Furthermore, the incubation period will allow the replication of the plasmid DNA (if it got in). This will enable the transformed cells to exhibit antibiotic resistance so that they will grow on your selective media plate
I have a question.
Do you guys grow your bacteria in LB or SOc medium wo antibiotics after you heat shock them (before plating). I remember my old supervisor saying that this was not required for amp. resist plasmids.But I do not remember why?
Would be nice to hear why and what you do.
Thanks
I haven't heard this before. bacteria need some time to recover and express its antibiotic resistence gene before you put them onto antibiotic containing plate. otherwise they would not survive. I always grow my bacteria in SOC medium which is richer in nutrients than LB. I generally grow them like 45 min or an hour before plating.
But if u r using the cloning kit (e.g. from qiagen or invitrogen), the growing period after heat shock is not necessary, why?
It is a short cut. But the cells still sit in SOC, thus they do recover in SOC medium for some time until all the media is absorbed by the plate agar. Ampicilin only kills the cells when they try to divide. After transformation the cells are a little worse for wear and thus pause for some time recovering before begining new round of cellular division.
Recovery in SOC is a requirement, especially with antibiotics that intefere with protein synthesis. If the bacteria where immediate plated on such an antibiotic laced agar , it would be the end of said bacteria, as it can't even produce the proteins to give it resistence.
We grow them for 40-60 min. in SOC or SOB w/o antibiotics before plating them. You could grow them in LB as well.
Perneseblue has explained why its necessary for recovery.
While cells recover in 1 hour for kanamycin resistance, we find that 2 hours increases transformation efficiency significantly for tetracycline or chloramphenicol resistant plasmids. Amp, as mentioned above, needs little or no recovery time.
kanamycin kills all cells, ampicillin kills only growing, dividing cells. So some companies now have '5 minute transformation' protocols, but for ampicillin only. However, it's not all that efficient, so unless your transformation is normally really reliable and you just want to save some time, just stick with the hour-long incubation in SOc, with no antibiotics.
This is a good document to read btw - http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/PDF/TransSG.pdf © Dean Madden