I am freezing a strain of cells, DH5 alpha, that contain a plasmid, which I call "7". It is about 5200bp, and holds the gene for RFP. I inoculated 200µl of an overnight liquid culture into a centrifuge tube, then took 50µl of 80% glycerol and put that into the tube as well (variable amount of glycerol here. the example is for my 10% batch, I also a 20% and 40%). I then vortexed the cells, and put one batch into the -80 and one into the -20
-- I except the -80 cells to stay good for at least 6 months, but I am not sure about the -20s. I hope at least 3 months, because then I could begin storing strains with my plasmids at my home lab, which will significantly help me since I've found fresh DNA to usually be better than old DNA in some of my experiments. Plus I can directly put that onto plates or liquid culture to send to people.
-Koeng
On Friday, June 27, 2014 4:11:39 PM UTC-7, scriptdoc wrote:
-Koeng
On Friday, June 27, 2014 4:11:39 PM UTC-7, scriptdoc wrote:
I think that it is not the freezing but the thawing out that is hard on cells. No idea how long your cells will last because different cells have different life spans. I would like to know more of what you are doing, the type of cells used, collection methods and your reasoning or expected outcome.thank youOn Fri, Jun 27, 2014 at 4:06 PM, Koeng <koen...@gmail.com> wrote:
Hey--I wanted to try out freezing cells in a -20 freezer at 10% glycerol a few days ago. So I did it, and it looks like they are frozen solid. I don't know much about freezing cells, but I assume since they are solid they should stay good for longer. I forgot to check the ones with 40% glycerol, but perhaps they aren't frozenHas anyone tried this? Does anyone know if cells that are frozen solid will degrade over a long period of time? Of course I am trying it, I have cells in 10% 20% and 40% glycerol in both -20 and -80, but I'd like to know if anyone has experience with this. In a month or so I'll try to recover some plasmid from them. I'll just keep trying each couple of months and if it's working I'll start doing it ever 3 months. I am putting it directly into liquid culture, so all I am testing is if there is still plasmid, so I don't know how useful the results will beAnyone have any experience with this? (BTW NEB says that their comp cells become less competent in -20, but did they freeze them solid? Is there a difference? Sorry, I am no expert at this, so that's why I am trying it)-Koeng
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