Hi everyone,
We have some liquid cultures growing that we would like to use for both making long-term glyercol stocks and for purifying plasmids from them. In between the liquid culture being ready and us purifying the plasmids there will be almost a week of time so I was wondering if I can just freeze the liquid culture and use that later for the plasmid purification instead of having to regrow glyercol stocks.
As I see it, freezing in medium will only damage the cells but as they will be lysed anyways during the purification I assume that should not matter...
I would make the glycerol stock and then freeze pellet(s) for the plasmid purification. For the plasmid prep, take out the frozen pellet (with no liquid) and add the resuspension buffer to it. Swirl on an orbit shaker slowly until the pellet is thawed and resuspended. You may have to use a pipette or vortex to get a single cell suspension. You could also thaw the pellet before adding the buffer, but the former method is faster.
This is our routine and we store pellets for weeks until we need the plasmid. Freezing the liquid would be less desirable since some cells would lyse during the freeze/thaw and you would lose plasmid. I guess if you made a large glycerol stock of your culture for plasmid then that would work too. With freezing the pellets, you're already one step ahead on the plasmid prep day.
I would make the glycerol stock and then freeze pellet(s) for the plasmid purification. For the plasmid prep, take out the frozen pellet (with no liquid) and add the resuspension buffer to it. Swirl on an orbit shaker slowly until the pellet is thawed and resuspended. You may have to use a pipette or vortex to get a single cell suspension. You could also thaw the pellet before adding the buffer, but the former method is faster.
This is our routine and we store pellets for weeks until we need the plasmid. Freezing the liquid would be less desirable since some cells would lyse during the freeze/thaw and you would lose plasmid. I guess if you made a large glycerol stock of your culture for plasmid then that would work too. With freezing the pellets, you're already one step ahead on the plasmid prep day.
I agree with Christopher, no reason to freeze the liquid culture and you are likely to get loss. Just freeze the pellet and then gently warm and proceed. Be aware though that you are likely to get significant lysis of bacterial cells once you resuspend in your first buffer.
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