I am facing problems with NEB Stable Cells. The original stock has an expiry date of 2020 but we thought the glycerol stocks would be fine. We are not able to clone and transform in NEB competent cells. For control we also cloned in DH5-alpha, and we were able to clone in that.
Specifically, our NEB Stable cells have lost their re-growth potential. They form few colonies in the first plate but when confirmed for positive clones and streaked on second plate they show no growth. Can somebody help?
Depending upon how they were stored and the number of freeze thaw cycles they have undergone, stock competent cells do have a limited life time in the freezer, although specifying how long is difficult due to the above. If you are no longer getting good transformants, you might just restreak a bit directly on LB or similar plates from the frozen stock and then prepare new competent cells from that, if you can't purchase a new stock.
Thank you Michael for the answer. We have re-streaked and re-innoculated on agar plates and broth. But still we are facing the same issues. Anyway, we have purchased new stock for now, but I was just curious to know what is the factor that is causing these bacteria to suddenly stop growing in the media. I mean do these companies do something so that they have a limited shelf life?
There is nothing that should stop the strain from growing. The limited shelf life is only for efficient competence. But once you have streaked it out, then the strain should grow fine. So I am not sure why you would be having problems unless you have some sort of phage contamination or similar.
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