I have an 11 year old glycerol stock of a coat protein that is in pET21(d)+ expression vector and transformed with BL21-(DE3) transformation cells that will not grow on my LB agar w /ampicillin plate. The vial has been stored for the last 11 years in a -80* freezer and never really taken out and worked with until now. I am now trying to recover the protein from the glycerol stock but like I said, I'm having no luck getting it to grow. I'm pretty sure if the coat protein was viable going into the -80 then even after 11 years it should be viable coming out. Does anybody have any suggestions of things I could try other than growing it on a LB agar w/ amp. plate to see if there is anything actual in the glycerol stock? Would/could the protein actually degrade that much over time in a -80* freezer? I was not at my current job 11 years ago so unfortunately I don't have a lot of knowledge of what may have been done with this culture in the past. Any help or suggestions would be greatly appreciated, thanks.
True, a well-made glycerol stock should last 11 years, but you have no control over what might had happened in the intervening period. The stock might have been defrosted repeatedly, or perhaps it was not very well prepared to begin with -stocks prepared from overnight cultures tend not to last that long, for instance.
If I were you I'd try rescuing the plasmid by taking an aliquot, spinning it to get a cell pellet, resuspending the pellet in water and boiling for 5 min, then spinning again and using the supernatant to transform high efficiency competent cells.
We had the similar problem once, we tried the LB plate first it never worked, so we just added a small amount of glycerol stock into LB media with the specific antibiotic.Luckily, the small amount of viable cells in the stock grow in the overnight culture. You can try the same approach to see it works.
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