Cell culture - (Aug/22/2007 )
Hi, everybody,
I am new here and just knew this useful forum recently. I learned a lot here.
I am struggling with growing E. coli. I start overnight culture with glycerol stocks (I used this stock a few month ago, and it was OK). It is so strange that the overnight culture is usually OK, but when i innoculate into 1L medium, cells start to die. I don't know if it is because i used the flasks which have been used to grow yeast. Would somebody kindly tell me the reason about it or give me some suggestions. Thanks a ton in advance! BOW!
I am new here and just knew this useful forum recently. I learned a lot here.
I am struggling with growing E. coli. I start overnight culture with glycerol stocks (I used this stock a few month ago, and it was OK). It is so strange that the overnight culture is usually OK, but when i innoculate into 1L medium, cells start to die. I don't know if it is because i used the flasks which have been used to grow yeast. Would somebody kindly tell me the reason about it or give me some suggestions. Thanks a ton in advance! BOW!
Hi,
Growing E. coli shouldn't be a problem even if you used that kind of flask bcoz I'm sure it was cleaned and sterilized prior to usage.But, what strain of E.coli are you using? If it was for cloning work, there's a several strain wif a special feature where they require a selective medium (usually mixed wif certain antibiotics).
Since you had been using the same stock, then, there might be two possibilities, something wrong wif your media or your stock had been contaminated. Why don't you prepare a new media and make sure everything is in optimal condition, then inoculate the same stock. At the same time, compare it wif another set of stock (which you never used. I'm sure you have a lots of aliquots). If they grow together, then something to do wif your media. If the old one died, then the stock had been contaminated or mutated and a lot more possibilites.
I used to have the same problem, later I found out, it the medium. Try to have a look on the yeast extract composition of your media. I'm not sure how muc this can help. Anyway, good luck.
Hi, p38, thank you so so so so ...... much for your reply!
The E. coli strain we are using is BL21(DE3)pJY2 and our enzyme is in pET-3d vector. So, I put antibiotics Ampicilin (final 0.2mg/ml) and chloramphenical (0.06mg/ml) in the 2xyt media from sigma (we always use this media). I usually prepare new media just the day before i grow cells to cool down. It worked when i grew cells from the glycerol stock a few month ago. It also worked when others grew them before. We only have two stock left and I tried both. None of them worked.
Strangely, the overnight culture was OK. Does this mean the glycerol stock is fine? Even stranger, for some other protein (not the one i am growing recently), when I inoculate for the first time into 1L media, cells could not grow. Three hrs later when i realized cells died, I added more overnight culture (from different flask, but from the same stock) into the same media in the same 1L flask, cells grew well.
I am not sure if my glycerol stock got contaminated. Is it a good idea to streak out the glycerol stock onto plates and make new stocks?
Thanks a million!
yu